Ionic polymers as toxin-binding agents

ABSTRACT

A method for treating pathogenic toxins in a mammal, such as a human, comprising treating the mammal with a therapeutically effective amount of a polymer comprising a cationic group attached to the polymer backbone. The polymer can be a homopolymer or a copolymer. In one embodiment, the polymer is a copolymer comprising a monomer having a pendant ammonium group and a hydrophobic monomer.

RELATED APPLICATION

[0001] This application is a Continuation of 09/597,343, filed Jun. 19,2000, which is a Continuation-in-Part of 09/412,474, filed Oct. 5, 1999,which is a Continuation of 08/934,495, filed Sep., 19, 1997, now U.S.Pat. No.: 6,007, 803, the entire teachings of which are incorporatedherein by reference.

BACKGROUND OF THE INVENTION

[0002] Many pathogens produce toxins which are detrimental, and in somecases, lethal, to the host organism. Toxins produced by pathogens can besecreted, or excreted from pathogenic organisms (e.g., “exotoxins”) ortoxic structural elements of pathogenic organisms (e.g., “endotoxins,”or toxin structural proteins).

[0003] Exotoxins are generally proteins or polypeptides. These toxins,which are secreted by the pathogen, can travel within the host and causedamage in regions of the host far removed from the infection site.Symptoms associated with exotoxins vary greatly and include hemolysis,systemic shock, destruction of leukocytes, vomiting, paralysis anddiarrhea.

[0004] Enterotoxins are exotoxins which act on the small intestine andcause massive secretion of fluid into the intestinal lumen, leading todiarrhea. Enterotoxins are produced by a variety of bacteria andviruses, including the food-poisoning organisms Staphylococcus aureus,Clostridium perfringens, and Bacillus cereus, and the intestinalpathogens Vibrio cholerae, Escherichia coli, and Salmonella enteritidis.

[0005] Endotoxins are lipopolysaccharides/lipoproteins found in theouter layer of the cell walls of gram-negative bacteria. Theselipopolysaccharides are bound to the cell membrane and are released uponcytolysis. Symptoms associated with the release of endotoxins includefever, diarrhea and vomiting. Specifically, endotoxins stimulate hostcells to release proteins, endogenous pyrogens, which affect the area ofthe brain which regulates body temperature. In addition to fever,diarrhea and vomiting, the host animal may experience a rapid decreasein lymphocyte, leukocyte, and platelet numbers, and enter into a generalinflammatory state.

[0006] Although endotoxins are less toxic than exotoxins, large doses ofendotoxins can cause death, generally through hemorrhagic shock andtissue necrosis. Examples of bacteria which produce endotoxins includethe genera Escherichia, Shigella, and especially Salmonella.

[0007] In some cases, the active disease caused by an exotoxin can betreated by administering an antitoxin to the patient. An antitoxincomprises antibodies to the toxin derived from the serum of an animal,typically a horse, which has been immunized by injection of a toxoid, anontoxic derivative of the toxin. However, the effectiveness ofantitoxins is limited because toxins are rapidly taken up by cells andbecome unavailable to the antibodies. Furthermore, the patient's immunesystem can respond to foreign proteins present in the antitoxin,creating a condition known as serum sickness.

[0008] Therefore, a need exists for an improved method of treatingtoxins which significantly reduces or eliminates the above-mentionedproblems.

SUMMARY OF THE INVENTION

[0009] One aspect of the present invention is a method for inhibiting apathogenic toxin in a mammal, comprising administering to the mammal atherapeutically effective amount of a polymer having a cationic group,such as an amino group, an ammonium group or a phosphonium group, whichis connected to the polymer backbone.

[0010] The polymer to be administered can be a homopolymer or acopolymer. In one embodiment, the polymer further includes a monomercomprising a hydrophobic group, such as an aryl group or a normal orbranched C₂-C₂₄-alkyl group.

[0011] The polymer to be administered can, optionally, further include amonomer comprising a neutral hydrophilic group, such as a hydroxyl groupor an amide group.

[0012] Another aspect of the invention is a method for inhibiting apathogenic toxin in a mammal, such as a human, comprising administeringto the mammal a therapeutically effective amount of a polymer comprisinga polymethylene backbone which is interrupted at one or more points by acationic group, such as an amino group, an ammonium group or aphosphonium group.

[0013] The present method has several advantages. For example, thepolymers employed are easily prepared using standard techniques ofpolymer synthesis and inexpensive starting materials. The polymers willnot be substantially degraded in the digestive tract and, therefore, canbe administered orally. Polymer compositions can also be readily varied,to optimize properties such as solubility or water swellability andantitoxin activity.

DETAILED DESCRIPTION OF THE INVENTION

[0014] A description of preferred embodiments of the invention follows.

[0015] The present invention relates to a method for inhibiting apathogenic toxin in a mammal, such as a human, by administering to themammal a therapeutically effective amount of a polymer comprising aplurality of amino or ammonium groups.

[0016] As used herein, the inhibition of a pathogenic toxin refers tothe reduction in activity of a toxin produced by a pathogenic microbe.The activity of the toxin can be reduced, for example, by interferingwith the production or secretion of the toxin or by binding the toxin toform an inactive complex. Without being bound by theory, one mechanismby which the polymers disclosed herein may inhibit a pathogenic toxin isby binding the toxin.

[0017] A “therapeutically effective amount” is an amount sufficient toinhibit, partially or totally, the activity of a pathogenic toxin. Theterm “polymer” refers to a macromolecule comprising a plurality ofrepeat units or monomers. The term includes homopolymers, which areformed from a single type of monomer, and copolymers, which are formedof two or more different monomers. A “terpolymer” is a copolymer formedfrom three different monomers. The term polymer, as used herein, isintended to exclude proteins, peptides, polypeptides and proteinaceousmaterials.

[0018] As used herein, the term “polymer backbone” or “backbone” refersto that portion of the polymer which is a continuous chain, comprisingthe bonds which are formed between monomers upon polymerization. Thecomposition of the polymer backbone can be described in terms of theidentity of the monomers from which it is formed, without regard to thecomposition of branches, or side chains, off of the polymer backbone.Thus, poly(acrylamide) is said to have a poly(ethylene) backbonesubstituted with carboxamide (—C(O)NH₂) groups as side chains.

[0019] The term “polymer side chain” or “side chain” refers to theportion of a monomer which, following polymerization, forms a branch offof the polymer backbone. In a homopolymer, all of the polymer sidechains are identical. A copolymer can comprise two or more distinct sidechains. When a side chain comprises an ionic unit, for example, theionic unit depends from, or is a substituent of, the polymer backbone,and is referred to as a “pendant ionic unit”. The term “spacer group”,as used herein, refers to a polyvalent molecular fragment which is acomponent of a polymer side chain and connects a pendant moiety to thepolymer backbone. The term “aliphatic spacer group” refers to a spacergroup which does not include an aromatic unit, such as a phenylene unit.

[0020] The term “addition polymer”, as used herein, is a polymer formedby the addition of monomers without the consequent release of a smallmolecule. A common type of addition polymer is formed by polymerizingolefinic monomers, wherein monomers are joined by the formation of acarbon—carbon bonds between monomers, without the loss of any atomswhich compose the unreacted monomers.

[0021] The term “monomer”, as used herein, refers to both (a) a singlemolecule comprising one or more polymerizable functional groups prior toor following polymerization, and (b) a repeat unit of a polymer. Anunpolymerized monomer capable of addition polymerization, can, forexample, comprise an olefinic bond which is lost upon polymerization.

[0022] The term “cationic group”, as used herein, refers to a functionalgroup which bears a net positive charge or a basic group which gains anet positive charge upon protonation at physiological pH. Suitablecationic groups include ammonium groups, such as primary, secondary,tertiary and quaternary ammonium groups; amino groups, such as primary,secondary and tertiary amino groups; sulfonium groups; and phosphoniumgroups.

[0023] The quantity of a given polymer to be administered will bedetermined on an individual basis and will be determined, at least inpart, by consideration of the individual's size, the severity ofsymptoms to be treated and the result sought. The polymer can beadministered alone or in a pharmaceutical composition comprising thepolymer, an acceptable carrier or diluent and, optionally, one or moreadditional drugs.

[0024] The polymers can be administered, for example, topically, orally,intranasally, or rectally. The form in which the agent is administered,for example, powder, tablet, capsule, solution, or emulsion, depends inpart on the route by which it is administered. The therapeuticallyeffective amount can be administered in a series of doses separated byappropriate time intervals, such as hours.

[0025] Pathogenic toxins which can be inhibited by the method of thepresent invention include, but are not limited to, toxins produced by amicroorganism, such as bacteria, viruses, protozoa, fungi or parasites.Such toxins include bacterial toxins, such as those produced byStreptococcus, including Streptococcus pneumoniae, and Streptococcuspyogenes; Salmonella, including Salmonella enteritidis; Campylobacter,including Campylobacter jejuni; Escherichia coli; Clostridia, includingClostridium difficile and Clostridium botulinum; Staphylococcus,including Staphylococcus aureus; Shigella dysenteriae; Pseudomonasincluding Pseudomonas aeruginosa; Bordatella pertussis; Listeriamonocytogenes; Vibrio cholerae; Yersinia enterocolitica; Legionellapneumophilia; and Bacillus anthracis.

[0026] Of particular pathogenic importance are Escherichia coli, forexample, E. coli strains 06:H—, 0157:H7,0143 and other clinicalisolates, and Clostridium difficile. Enterohemorrhagic Esherichia coli(EHEC), such as 0157:H7, can cause a characteristic nonfebrile bloodydiarrhea known as hemorrhagic colitis. EHEC produce high levels of oneor both of two related cytotoxins which resemble a Shiga toxin instructure and function and are referred to as Shiga-like toxins (SLT Ior SLT II). These Shiga toxins are believed to damage the intestinalmucosa, resulting in the manifestation of hemorrhagic colitis.

[0027]Clostridium difficile produce two major toxins, designated Toxin Aand Toxin B, which cause damage to the cellular lining of the bowelwall. Toxin A causes fluid production and damage to the mucosa of thelarge bowel. Toxin B is a cytotoxin which causes abnormalities in tissueculture systems. This quality of Toxin B is used to diagnose the diseaseby detecting toxin in feces.

[0028] Also included are protozoal toxins, such as toxins produced byEntameoba histolytica, and Acanthameoba; and parasitic toxins.

[0029] The method of the invention can also be used to inhibit a viraltoxin, such as a toxin produced by rotavirus, human immunodeficiencyvirus, influenza virus, polio virus, vesicular stomatitis virus,vaccinia virus, adenovirus, picomavirus, togaviruses (such as sindbisand semlikifores viruses), paramyxoviruses, papillomaviruses. Toxinswhich can be inhibited using the method of the invention includeviroporin molecules produced by any of these viruses. A preferred toxinwhich can be inhibited using the method of the invention is therotavirus NSP4 protein. Other toxins which can be inhibited includeinfluenza M2 protein, HIV Vpu and gp41 proteins, picomavirus 3A protein,togavirus 6K protein, respiratory syncitial virus SH protein,coronavirus D3 protein and adenovirus E5 protein.

[0030] The method is useful for treating infections of various organs ofthe body, but is particularly useful for infections of the skin andgastrointestinal tract.

[0031] Polymers which are particularly suitable for the present methodinclude polymers which can possess key characteristics of naturallyoccurring antigens, in particular, the ability to form amphipathicstructures. The term “amphipathic”, as used herein, describes athree-dimensional structure having discrete hydrophobic and hydrophilicregions. Thus, one portion of the structure interacts favorably withaqueous and other polar media, while another portion of the structureinteracts favorably with non-polar media. An amphipathic polymer resultsfrom the presence of both hydrophilic and hydrophobic structuralelements along the polymer backbone.

[0032] Polymers to be administered which have amino groups can beadministered in the free base, amino form, or as a salt with apharmaceutically acceptable acid. Such acids include hydrochloric acid,hydrobromic acid, citric acid, lactic acid, tartaric acid, phosphoricacid, methanesulfonic acid, acetic acid, formic acid, maleic acid,fumaric acid, malic acid, succinic acid, malonic acid, sulfuiric acid,L-glutamic acid, L-aspartic acid, pyruvic acid, mucic acid, benzoicacid, glucoronic acid, oxalic acid, ascorbic acid, and acetylglycine. Ineither case, at physiological pH following administration, a pluralityof amino groups will be protonated to become ammonium groups, and thepolymer will carry an overall positive charge.

[0033] Polymers comprising quaternary ammonium groups will furthercomprise a pharmaceutically acceptable counter anion, such as an anionwhich is a conjugate base of one of the pharmaceutically acceptableacids discussed above. The number of counter anions associated with thepolymer prior to administration is the number necessary to balance thepositive charge on the polymer.

[0034] The polymer to be administered can be an addition polymer havinga polymer backbone such as a polyacrylate, polyacrylamide,poly(allylalcohol), poly(vinylalcohol), poly(vinylamine),poly(allylamine), or poly(diallylamine) backbone. The polymer can have auniform backbone if it is composed of monomers derived from a commonpolymerizable unit, such as acrylamide. If the polymer is a copolymer,it can also comprise a mixed backbone, a block copolymer backbone, agrafted backbone or an interpenetrating polymer backbone.

[0035] The polymers of use in the present method also includecondensation polymers, wherein polymerization of monomers is accompaniedby the release of a small molecule, such as a water molecule. Suchpolymers include, for example, polyesters and polyurethanes.

[0036] The polymers of use in the present method can be linear orcrosslinked. The polymer can be crosslinked, for example, by theincorporation within the polymer of a multifunctional comonomer.Suitable multifunctional co-monomers include diacrylates, triacrylatesand tetraacrylates, dimethacrylates, diacrylamides, diallylacrylamide,di(methacrylamides), triallylamine and tetraalylammoniumion. Specificexamples include ethylene glycol diacrylate, propylene glycoldiacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate,butylene glycol dimethacrylate, methylene bis(methacrylamide), ethylenebis(acrylamide), ethylene bis(methacrylamide), ethylidenebis(acrylamide), ethylidene bis(methacrylamide), pentaerythritoltetraacrylate, trimethylolpropane triacrylate, bisphenol Adimethacrylate, and bisphenol A diacrylate. Other suitablemultifunctional monomers include polyvinylarenes, such asdivinylbenzene. The amount of crosslinking agent is typically betweenabout 1.0% and about 30% by weight relative to the weight of thepolymer, preferably from about 5% to about 25% by weight.

[0037] The polymer can also be crosslinked by bridging units which linkamino groups on adjacent polymer strands. Suitable bridging unitsinclude straight chain or branched, substituted or unsubstitutedalkylene groups, diacylalkylene groups, diacylarene groups and alkylenebis(carbamoyl) groups. Examples of suitable bridging units include—(CH₂)_(n)—, wherein n is an integer from about 2 to about 20;—CH₂—CH(OH)—CH₂—; —C(O)CH₂CH₂C(O)—;—CH₂—CH(OH)—O—(CH₂)_(m)—O—CH(OH)—CH₂—, wherein m is an integer fromabout 2 to about 4; —C(O)—(C₆H₂(COOH)₂)—C(O)— and—C(O)NH(CH₂)_(p)NHC(O)—, wherein p is an integer from about 2 to about20.

[0038] Advantageously, crosslinking the polymers renders the polymersnon-adsorbable and stable in the patient. A “stable” polymercomposition, when administered in therapeutically effective amounts,does not dissolve or otherwise decompose to form potentially harmfulbyproducts, and remains substantially intact.

[0039] The polymer can be crosslinked, for example, by including amultifunctional co-monomer as the crosslinking agent in the reactionmixture. A multifunctional co-monomer can be incorporated into two ormore growing polymer chains, thereby crosslinking the chains. Suitablemultifunctional co-monomers include those discussed above. The amount ofcrosslinking agent added to the reaction mixture is, generally, between1.0% and 30% by weight relative to the combined weight of the polymerand the crosslinking agent, and preferably from about 2.5% to about 25%by weight.

[0040] The multifunctional co-monomer can also take the form of amultifunctional diallylamine, such as a bis(diallylamino)alkane or abis(diallylalkylammonio) alkane. Suitable monomers of this type include1,10-bis(diallylmethylammonio)decane dibromide and1,6-bis(diallylmethylammonio)hexane dibromide, each of which can beformed by the reaction of diallylmethylamine with the appropriatedibromoalkane.

[0041] In one embodiment, the polymer to be administered comprises amonomer, or repeat unit, of Formula I,

[0042] wherein X is a covalent bond, a carbonyl group or a CH₂ group, Yis an oxygen atom, an NH group or a CH₂ group, Z is an spacer group, Ris a hydrogen atom or a methyl or ethyl group; R¹, R² and R³ are each,independently, a hydrogen atom, a normal or branched, substituted orunsubstituted C₁-C₂₄-alkyl group, an aryl group or an arylalkyl group;A⁻ is a pharmaceutically acceptable anion, such as a conjugate base of apharmaceutically acceptable acid; and m and n are each, independently, 0or 1. Suitable alkyl substituents include halogen atoms, such asfluorine or chlorine atoms. A monomer of Formula 1 in which at least oneof substituents R¹, R² and R³ is hydrogen can also exist in the freebase, or amino, form in which a hydrogen substituent is absent and thenitrogen atom is electrically neutral.

[0043] In a preferred embodiment, one of R¹-R³ is an ammonioalkyl groupof the general formula

[0044] wherein R⁴, R⁵ and R⁶ are each, independently, a hydrogen atom, aC₁-C₂₄ alkyl group, or an arylalkyl group; n is an integer from 2 toabout 20, preferably from 3 to about 6; and A⁻ is a pharmaceuticallyacceptable anion. An ammonioalkyl group in which at least one ofsubstituents R⁴, R⁵ and R⁶ is hydrogen can also exist in the free base,or amino, form in which a hydrogen substituent is absent and thenitrogen atom is electrically neutral. The group —N⁺(R⁴)(R⁵)(R⁶) canalso be a heteroaryl group, such as a 5- or 6-membered heteroaryl group,such as a 1-pyridinio group. Preferably, at least one of R⁴, R⁵ and R⁶is a C₆-C₂₄-alkyl group. Examples of suitable ammonioalkyl groupsinclude, but are not limited to,

[0045] 4-(dioctylmethylammonio)butyl; 3-(dodecyldimethylammonio)propyl;

[0046] 3-(octyldimethylammonio) propyl; 3-(decyldimethylammonio)propyl;

[0047] 5-(dodecyldimethylammonio)pentyl;3-(cyclohexyldimethylammonio)propyl;

[0048] 3-(decyldimethylammonio)-2-hydroxypropyl;3-(tridecylammonio)propyl;

[0049] 3-(docosyldimethylammonio)propyl;4-(dodecyldimethylammonio)butyl;

[0050] 3-(octadecyldimethylammonio)propyl;3-(hexyldimethylammonio)propyl;

[0051] 3-(methyldioctylammonio)propyl; 3-(didecylmethylammonio)propyl;

[0052] 3-(heptyldimethylammonio)propyl; 3-(dimethylnonylammonio)propyl;

[0053] 6-(dimethylundecylammonio)hexyl; 4-(heptyldimethylammonio)butyl;

[0054] 3-(dimethylundecylammonio)propyl;3-(tetradecyldimethylammonio)propyl

[0055] 3-(1-pyridinium)propyl; in combination with a pharmaceuticallyacceptable anion.

[0056] When at least one of R¹ to R⁶ is a hydrogen atom, the monomer canalso exist in the free base, or amino form. The polymer comprising sucha monomer can be administered in the free base form or in the protonatedform, for example, as a salt of a pharmaceutically acceptable acid.

[0057] The spacer group Z is a component of the polymer side chain andconnects the amino or ammonium group to the polymer backbone. The aminoor ammonium group is, thus, a pendant group. The spacer group can be anormal or branched, saturated or unsaturated, substituted orunsubstituted alkylene group, such as a polymethylene group —(CH₂)_(n)—,wherein n is an integer from about 2 to about 24. Suitable examplesinclude the propylene, hexylene and octylene groups. The alkylene groupcan also, optionally, be interrupted at one or more points by aheteroatom, such as an oxygen, nitrogen (e.g, NH) or sulfur atom.Examples include the oxaalkylene groups —(CH₂)₂O[(CH₂)₂O]_(n)(CH₂)₂—,wherein n is an integer ranging from 0 to about 3.

[0058] Examples of monomers of Formula I having quaternary ammoniumgroups include:

[0059] N-(3-dimethylaminopropyl)acrylamide,

[0060] N-(3-trimethylammoniopropyl)acrylamide,

[0061] 2-trimethylammonioethyl methacrylate,

[0062] 2-trimethylammonioethyl acrylate,

[0063] N-(3-trimethylammoniopropyl)methacrylamide,

[0064] N-(6-trimethylammoniohexyl)acrylamide,

[0065] N-(3-trimethylammoniopropyl)acrylamide,

[0066] N-(4-trimethylammoniobutyl)allylamine,

[0067] N-(3-dimethyloctylammoniopropyl)allylamine,

[0068] N-(3-trimethylammoniopropyl)allylamine,

[0069] N-(3-(1-pyridinio)propyl)vinylamine and

[0070] N-(3-(1-pyridinio)propyl)allylamine.

[0071] Each of these monomers also includes a suitable counter anion.Examples of monomers of Formula I having an amino group includeallylamine, vinylamine and N-(3-dimethylamino-propyl)acrylamide. Each ofthese monomers can also exist as a salt with a pharmaceuticallyacceptable acid.

[0072] In one embodiment, the repeat unit of Formula 1 is of the formula

[0073] where R₁, R₂, R₃ and A- have the meanings given above for formulaI. In a preferred embodiment, R₁, R₂ and R₃ are each hydrogen. Forexample, the polymer can be polyallylamine which is protonated on atleast a portion of the nitrogen atoms. In a preferred embodiment, thepolymer is protonated polyallylamine which is cross-linked with adifunctional cross-linking agent as described above. For example, theprotonated polyallylamine can be cross-linked with an epihalohydrin,such as epichlorohydrin. In a specific example, the polymer to beadministered is poly(allylamine) hydrochloride cross-linked with 5 to10% by weight epichlorohydrin.

[0074] In another embodiment, the polymer to be administered ischaracterized by a diallylamine repeat unit of Formula III:

[0075] wherein R¹ and R² are each, independently, a hydrogen atom, anormal or branched, substituted or unsubstituted C₁-C₂₄-alkyl group, anaryl group or an arylalkyl group; and A⁻ is a pharmaceuticallyacceptable anion, such as a conjugate base of a pharmaceuticallyacceptable acid. Suitable alkyl substituents include halogen atoms, suchas fluorine or chlorine atoms. A monomer of Formula III in which atleast one of substituents R¹ and R² is hydrogen can also exist in thefree base, or amino, form, in which a hydrogen substituent is absent andthe nitrogen atom is electrically neutral. In a preferred embodiment, R¹is an ammonioalkyl group of Formula II, as described above.

[0076] In another embodiment, the polymer to be administered is acopolymer characterized by a first repeat unit of Formula III whereinboth R¹ and R² are hydrogen and a second repeat unit of Formula IIIwherein R¹ and R² are each, independently, a C₁-C₂₄-alkyl group.Preferably, in the second repeat unit of Formula III, R¹ is a methylgroup and R² is a linear or branched C₁-C₁₈ alkyl group. The polymer canbe linear or cross-linked, as described above, and preferably includesfrom about 0.5 to about 20% by weight of a cross-linking agent, such asepichlorohydrin or one of the other difunctional cross-linking agentsdescribed above.

[0077] In another embodiment, the polymer to be administered is apoly(alkyleneimine) polymer comprising a monomer, or repeat unit, ofFormula IV,

[0078] wherein n is an integer from about 2 to about 10 and R⁷ and R⁸are each, independently, a hydrogen atom, a normal or branched,substituted or unsubstituted C₁-C₂₄-alkyl group, an aryl group or anarylalkyl group, and A⁻ is a pharmaceutically acceptable anion. Suitablealkyl substituents include halogen atoms, such as fluorine or chlorineatoms. When one of R⁷ and R⁸ is a hydrogen atom, the polymer can beadministered in the free base form or in the cationic form shown, as thesalt of a pharmaceutically acceptable acid. A monomer of Formula IV inwhich at least one of substituents R⁷ and R⁸ is hydrogen can also existin the free base, or amino, form, in which a hydrogen substituent isabsent and the nitrogen atom is electrically neutral. In a preferredembodiment, the polymer to be administered is a poly(ethyleneimine)polymer, comprising a monomer of Formula IV wherein n is 2.

[0079] Preferably, R⁷ is an aminoalkyl group, or an ammonioalkyl groupof Formula II, as described above. In one embodiment, the polymercomprises monomeric units of Formula II wherein R⁷ is an aminoalkylgroup, or an ammonioalkyl group, as well as monomeric units wherein R⁷and R⁸ are each hydrogen or R⁷ is hydrogen and R⁸ is absent. Thefraction of monomeric units which include the aminoalkyl or ammonioalkylgroup can be from about 5% to about 90% of the monomeric units of thepolymer.

[0080] Suitable polymers comprising a monomer of Formula II includepoly(decamethylenedimethylammonium-co-ethylenedimethylammonium) X⁻,wherein X⁻ is an anion, for example chloride or bromide;poly(ethyleneimine-co-N-decylethyleneimine-co-N-(trimethylammonio-propyl)ethyleneimine; poly(ethyleneimine-co-N-benzylethyleneimine).

[0081] The polymer to be administered can also be a copolymer comprisinga monomer of Formula I, Formula III or Formula IV and further comprisinga hydrophobic monomer. The hydrophobic monomer can comprise a side chainbearing a hydrophobic group, such as a straight chain or branched,substituted or unsubstituted C₃-C₂₄-alkyl group or a substituted orunsubstituted aryl group. Examples of suitable hydrophobic monomersinclude styrene, N-isopropylacrylamide, N-t-butylacrylamide,N-n-butylacrylamide, heptafluorobutyl acrylate, N-n-decylallylamine,N-n-decylacrylamide, pentafluorostyrene, n-butyl acrylate, t-butylacrylate, n-decyl acrylate, N-t-butylmethacrylamide, n-decylmethacrylate, n-butyl methacrylate, n-hexyl methacrylate,N-n-hexylvinylamine, N-n-hexylallylamine, N-benzylallylamine,N-(cyclohexylmethyl)allylamine, N-(n-decyl)allylamine,N-hexylethyleneimine, N-(3-phenylpropyl)ethyleneimine,N-decylethyleneimine and N-benzylethyleneimine.

[0082] Examples of copolymers characterized by a monomer of Formula Iand a hydrophobic monomer includepoly(N-(3-dimethylaminopropyl)acrylamide-co-N-n-butylacrylamide) orsalts thereof with pharmaceutically acceptable acids. Other examples ofsuitable copolymers includepoly(2-trimethylammonioethylmethacrylate-co-styrene) chloride,poly(2-trimethylammonioethylmethacrylate-co-N-isopropylacrylamide)chloride,poly(2-trimethyl-ammonioethylmethacrylate-co-heptafluorobutylacryl)chloride, poly(3-trimethylammoniopropylmethacrylate-co-styrene)chloride,poly(3-trimethylammonium-propylmethacrylate-co-N-t-butylacrylamide)chloride,poly(3-trimethylammoniopropylmethacrylate-co-N-n-butylacrylamide)chloride, andpoly(N-(3-trimethylammoniopropyl)allylamine-co-N-n-decylallylamine).Each of these ionic copolymers can also be employed with one or morecounter anions other than chloride, for example, with a conjugate baseof one or more pharmaceutically acceptable acids.

[0083] In a further embodiment, the polymer to be administered comprisesa monomer of Formula I, Formula III or Formula IV, a hydrophobic monomerand a neutral hydrophilic monomer, such as acrylamide, methacrylamide,N-(2-hydroxyethyl) acrylamide or 2-hydroxyethylmethacrylate. Examples ofpolymers of this type include terpolymers ofN-(3-trimethylammonium-propyl)methacrylamide/N-isopropylacrylamide/2-hydroxyethyl-methacrylate,N-(3-trimethylammonium-propyl)methacrylamide/N-n-decylacrylamide/2-hydroxyethylmethacrylate,N-(3-trimethyl-ammoniopropyl)methacrylamide/N-t-butylmethacrylamide/methacrylamide,N-(3-trimethylammonium-propyl)methacrylamide/n-decylacrylate/methacrylamide,2-trimethylammonioethylmethacrylate/n-butyl-acrylate/acrylamide,2-trimethyl-ammonium-ethylmethacrylate/t-butylacrylate/acrylamide,2-trimethylammonioethyl-methacrylate/n-decyl-acrylate/acrylamide,2-trimethylammonium-ethylmethacrylate/n-decylmethacrylate/methacrylamide,2-trimethylammonioethylmethacrylate/N-t-butyl-methacrylamide/methacrylamideand2-trimethylammonioethylmethacrylate/N-n-butyl-methacrylamide/methacrylamide.

[0084] In one embodiment, the polymer to be administered is across-linked polymer characterized by two or more monomers of Formula Iand/or Formula II. Preferably, the cross-linked polymer is characterizedby a first monomer having primary or secondary amino groups and a secondmonomer having tertiary amino groups or quaternary ammonium groups.Suitable examples of the first monomer include, but are not limited to,allylamine, vinylamine, N-alkylallylamine, N-alkylvinylamine anddiallylamine. Suitable examples of the second monomer include, but arenot limited to, N-alkyldiallylamine, N,N-dialkylallylamonium A-,N,N-dialkylallylamine, N,N,N-trialkylallylammonium A-. In the foregoingmonomers, A- is a suitable anion and the alkyl groups are preferablylinear or branched C₁-C₂₄-alkyl groups, more preferably C₁-C₄-alkylgroups, and most preferably methyl groups.

[0085] In one embodiment, the cross-linked polymer comprises two or morelinear polymers which are cross-linked as a mixture bypost-polymerization cross-linking. For example, the cross-linked polymercan be prepared by cross-linking two or more distinct linear polymers,that is, polymer strands having distinct chemical compositions..Preferably, the cross-linked polymer is prepared by cross-linking firstand second linear polymers having distinct compositions. In oneembodiment, the first linear polymer is characterized by a repeat unithaving a primary amino group and/or a repeat unit having a secondaryamino group and the second linear polymer is characterized by a repeatunit having a tertiary amino group and/or a repeat unit having aquaternary ammonium group. The second linear polymer can additionallyinclude one or more repeat units having primary and/or secondary aminogroups. Suitable examples for the first linear polymer includepolyallylamine, polyvinylamine, poly(ethyleneimine), polydiallylamine,N-alkylallylamine, for example, N-methylallylamine, andN-alkylvinylamine, for example, N-methylvinylamine. The second linearpolymer preferably includes repeat units having amino groups which willreact readily with a difunctional cross-linking agent, as describedabove. For example the second linear polymer preferably includes repeatunits having primary amino groups or repeat units having secondary aminogroups in addition to the repeat units having tertiary amino groups orquaternary ammonium groups. Suitable examples of the second linearpolymer include copolymers of N-alkyldiallylamine,N,N-dialkylallylamonium A-, N,N-dialkylallylamine, andN,N,N-trialkylallylammonium A- with at least one additional monomerwhich can react with a cross-linking agent. For example, the secondlinear polymer can be poly(N-alkyldiallylamine-co-diallylamine);poly(N,N-dialkyldiallylamonium-co-allylamine) A-;poly(N,N-dialkylallylamine-co-allylamine);poly(N,N-dialkylallylamine-co-N-alkylallylamine);poly(N,N,N-trialkylallylammonium-co-allylamine) A-;poly(N,N,N-trialkylallylammonium-co-N-alkylallylamine) A-;poly(N,N-dialkylvinylamine-co-vinylamine);poly(N,N-dialkylvinylamine-co-N-alkylvinylamine);poly(N,N,N-trialkylvinylammonium-co-vinylamine) A-; andpoly(N,N,N-trialkylvinylammonium-co-N-alkylvinylamine) A-.

[0086] The composition of the copolymers to be administered can varysubstantially. The copolymer can comprise from about 95 mole percent toabout 5 mole percent, preferably from about 20 mole percent to about 80mole percent, of a monomer of Formula I. The copolymer can also comprisefrom about 95 mole percent to about 5 mole percent, preferably fromabout 20 mole percent to about 80 mole percent, of a hydrophobicmonomer.

[0087] The polymers of use in the present method are preferablysubstantially nonbiodegradable and nonabsorbable. That is, the polymersdo not substantially break down under physiological conditions intofragments which are absorbable by body tissues. The polymers preferablyhave a nonhydrolyzable backbone, which is substantially inert underconditions encountered in the target region of the body, such as thegastrointestinal tract.

[0088] Other examples of polymers which are of use in the present methodare disclosed in U.S. patent application Ser. Nos. 08/482,969,08/258,477, 08/258,431, 08/469,659 and 08/471,769, the contents of eachof which are incorporated herein by reference.

[0089] The polymer to be administered will, preferably, be of amolecular weight which is suitable for the intended mode ofadministration and allows the polymer to reach and remain within thetargeted region of the body for a period of time sufficient to interactwith the toxin associated with the pathogen. For example, a method fortreating an intestinal infection should utilize a polymer ofsufficiently high molecular weight to resist absorption, partially orcompletely, from the gastrointestinal tract into other parts of thebody. The polymers can have molecular weights ranging from about 500Daltons to about 500,000 Daltons, preferably from about 2,000 Daltons toabout 150,000 Daltons.

[0090] The polymers which are useful in the present method can beprepared by known methods. A first method includes the directpolymerization of a monomer, such as trimethylammonioethylacrylatechloride, or a set of two or more monomers, such astrimethylammonioethylacrylate chloride, N-n-butylacrylamide andacrylamide. This can be accomplished via standard methods of freeradical, cationic or anionic polymerization which are well known in theart. Due to reactivity differences between two monomers, the compositionof a copolymer produced in this way can differ from the composition ofthe starting mixture. This reactivity difference can also result in anon-random distribution of monomers along the polymer chain.

[0091] A second method proceeds via the intermediacy of an activatedpolymer comprising labile side chains which are readily substituted by adesired side chain. An example of a suitable activated polymer is thesuccinimide ester of polyacrylic acid, poly(N-acryloyloxysuccinimide)(also referred to hereinafter as “pNAS”), which reacts with nucleophilessuch as a primary amine to form a N-substituted polyacrylamide. Anothersuitable activated polymer is poly(para-nitrophenylacrylate), whichreacts with amine nucleophiles in a similar fashion.

[0092] A copolymer having a polyacrylamide backbone comprising amidenitrogens bearing two different substituents can be prepared by treatingpNAS with less than one equivalent (relative to N-acryloyloxysuccinimidemonomer) of a first primary amine, producing a poly(N-substitutedacrylamide-co-N-acryoyloxysuccinimide) copolymer. RemainingN-acryoyloxysuccinimide monomer can then be reacted with, for example,an excess of a second primary amine to produce a polyacrylamidecopolymer having two different N-substituents. A variety of copolymercompositions can, thus, be obtained by treating the activated polymerwith different proportions of two or more amines.

[0093] Polymers suitable for use in the present method can also beprepared by addition of a side chain to a preformed polymer. Forexample, poly(ethyleneimine), poly(allylamine) and poly(vinylamine) caneach be alkylated at the amino nitrogen by one or more alkylatingagents. For example, a fraction of the amino groups can be alkylatedusing an alkylating agent such as a normal or branched C₃-C₂₄-alkylhalide, such as n-decyl bromide, or an (X-alkyl)ammonium salt, wherein Xrepresents a suitable leaving group, such as a halide, a tosylate or amesylate group. These compounds can be prepared by the reaction of anappropriate dihaloalkane, such as a bromochloroalkane, with a tertiaryamine. Suitable alkylating agents of this type include the following:

[0094] (4-bromobutyl)dioctylmethylammonium bromide;

[0095] (3-bromopropyl)dodecyldimethylammonium bromide;

[0096] (3-chloropropyl)dodecyldimethylammonium bromide;

[0097] (3-bromopropyl)octyldimethylammonium bromide;

[0098] (3-chloropropyl)octyldimethylammonium bromide;

[0099] (3-iodobutyl)dioctylmethylammonium bromide;

[0100] (2,3-epoxypropyl)decyldimethylammonium bromide;

[0101] (3-chloropropyl)decyldimethylammonium bromide;

[0102] (5-tosylpentyl)dodecyldimethylammonium bromide;

[0103] (6-bromohexyl)octyldimethylammonium bromide;

[0104] (12-bromododecyl)decyldimethylammonium bromide;

[0105] (3-bromopropyl)tridecylammonium bromide;

[0106] (3-bromopropyl)docosyldimethylammonium bromide;

[0107] (6-bromohexyl)docosyldimethylammonium bromide;

[0108] (4-chlorobutyl)dodecyldimethylammonium bromide;

[0109] (3-chloropropyl)octadecyldimethylammonium bromide;

[0110] (3-chloropropyl)hexyldimethylammonium bromide;

[0111] (3-chloropropyl)methyldioctylammonium bromide;

[0112] (3-chloropropyl)methyldidecylammonium bromide;

[0113] (3-chloropropyl)cyclohexyldimethylammonium bromide;

[0114] (3-bromopropyl)heptyldimethylammonium bromide;

[0115] (3-bromopropyl)dimethylnonylammonium bromide;

[0116] (6-bromohexyl)dimethylundecylammonium bromide;

[0117] (4-chlorobutyl)heptyldimethylammonium bromide;

[0118] (3-chloropropyl)dimethylundecylammonium bromide;

[0119] (3-chloropropyl)tetradecyldimethylammonium bromide; and

[0120] 1-(3-chloropropyl)pyridinium bromide.

[0121] Each of the alkylating agents described above can also beprepared and used as a salt in combination with an anion other thanbromide. For example, these and similar alkylating agents can beprepared and used as salts with a wide range of anions, includingchloride, iodide, acetate, p-toluenesulfonate and methanesulfonate.

[0122] When substituents are added to the polymer by way of analkylating agent as described above, the extent of alkylation can bedetermined by methods which are well known in the chemical arts. Theincrease in polymer mass due to alkylation provides a measure of theextent of alkylation. For example, in a reaction betweenpoly(allylamine) and 1-bromohexane, a product/starting polymer massratio of about 3.9, 2.5 and 1.7 represent approximately 100%, 50% and25% alkylation, respectively. The degree of alkylation can also bedetermined by elemental analysis of the product polymer. In this case,the carbon/nitrogen (C/N) mass ratio is a direct measure of the degreeof alkylation. For example, the reaction of poly(allylamine) with1-bromohexane yields a product with a higher C/N mass ratio than that ofthe starting polymer. Product C/N mass ratios of about 7.7, 5.1 and 3.9represent, approximately, 100%, 50% and 25% alkylation, respectively.

[0123] The polymer can be crosslinked, for example, by including amultifunctional co-monomer as the crosslinking agent in the reactionmixture. A multifunctional co-monomer can be incorporated into two ormore growing polymer chains, thereby crosslinking the chains. Suitablemultifunctional co-monomers include those discussed above. The amount ofcrosslinking agent added to the reaction mixture is, generally, between1.0% and 30% by weight relative to the combined weight of the polymerand the crosslinking agent, and preferably from about 2.5% to about 25%by weight.

[0124] The multifunctional co-monomer can also take the form of amultifunctional diallylamine, such as a bis(diallylamino)alkane or abis(diallylalkylammonio) alkane. Suitable monomers of this type include1,10-bis(diallylmethylammonio)decane dibromide and1,6-bis(diallylmethylammonio)hexane dibromide, each of which can beformed by the reaction of diallylmethylamine with the appropriatedibromoalkane.

[0125] The polymers to be administered can also be crosslinkedsubsequent to polymerization by reacting the polymer with one or morecrosslinking agents having two or more functional groups, such aselectrophilic groups, which react with amine groups to form a covalentbond. Crosslinking in this case can occur, for example, via nucleophilicattack of the polymer amino groups on the electrophilic groups. Thisresults in the formation of a bridging unit which links two or moreamino nitrogen atoms from different polymer strands. Suitablecrosslinking agents of this type include compounds having two or moregroups selected from among epoxide, acyl-X and alkyl-X, wherein X- is asuitable leaving group, such as a halide, acylate, tosylate or mesylategroup. Examples of such compounds include epichlorohydrin, succinyldichloride, butanedioldiglycidyl ether, ethanedioldiglycidyl ether,pyromellitic dianhydride and dihaloalkanes. The crosslinking agent canalso be an a,w-alkylene diisocyanate, for example OCN(CH₂)_(p)NCO,wherein p is an integer from about 2 to about 20. The polymer can bereacted with an amount of crosslinking agent equal to from about 0.5 to40 mole percent relative to the amino groups within the polymer,depending upon the extent of crosslinking desired.

[0126] As discussed below in Example 56, several polymers describedherein were tested for in vitro activity against Shiga toxins 1 and 2and exhibited excellent toxininhibiting properties.

[0127] The invention will now be further and specifically described bythe following examples.

EXAMPLES

[0128] The following abbreviations are used throughout the examples todenote the following monomers: MAPTAC,N-(3-trimethylammoniopropyl)methacrylamide chloride; TMAEMC,2-trimethylammonioethylmethacrylate chloride; HEMA,2-hydroxyethylmethacrylate; TMAEAC, 2-trimethylammonioethylacrylatechloride.

[0129] The copolymers and terpolymers of the following examples aregiven nominal compositions which correspond to the molar ratios ofstarting monomers in the copolymerization mixture.

Example 1 Synthesis of poly(N-acryloyloxysuccinimide) (PNAS)

[0130] A solution of N-acryloyloxysuccinimide (25.0 g, 148 mmole) in 100mL dry DMF was degassed by nitrogen purging and simultaneously heated to60° C. To the reaction mixture was added azobisisobutyronitrile (AIBN)(120 mg, 0.005 equivalents with respect to monomer). The reaction wasallowed to proceed for 24 hours at 60° C. The polymer solution wascooled to room temperature and poured into rapidly stirred THF. Theresulting white precipitate was filtered, washed with THF and dried invacuo.

Example 2 Synthesis ofpoly(N-(3-dimethylamino-propyl)acrylamide-co-N-n-butylacrylamide)

[0131] To a solution of 3.0 g (17.75 mmole) pNAS in 20 mL dry DMF wasadded 0.6 g (3.55 mmole) n-butylamine. The resulting solution wasstirred at room temperature for 14 hours, and then heated at 60° C. for4 hours. After the solution was cooled to room temperature, 9.05 g (89mmole) 3-dimethylaminopropylamine was added, and the resulting solutionwas stirred at room temperature for 2 hours, then heated to 60° C. for20 hours. After cooling to room temperature, the solution was dilutedwith 25 mL water, and dialyzed against water for 24 hours. The solutionwas then lyophilized to affordpoly(N-(3-dimethylaminopropyl-acrylamide)-co-N-n-butylacrylamide) as atacky white solid.

Example 3 Synthesis ofpoly(N-(3-trimethylammoniopropyl)acrylamide-co-N-n-butylacrylamide)iodide

[0132] To a suspension ofpoly(3-dimethylaminopropyl-acrylamide-co-N-n-butylacrylamide in methanolwas added 0.5 g methyl iodide. The resulting mixture was stirred for 3hours, and gradually became homogeneous. After stirring for another 12hours, the solvent was removed under reduced pressure and the polymerwas washed with dry hexane.

Example 4 Synthesis ofpoly(N-(2-hydroxyethyl)acrylamide-co-N-(6-trimethylammoniohexyl)acrylamide)bromide

[0133] To a solution of 2.48 g (15 mmole) pNAS in 5 mL DMF was added1.00 g (3 mmole) 1-trimethylammonium-6-hexanamine bromide. The solutionwas stirred at room temperature for 4 hours and then heated at 60° C.for 20 hours. The solution was cooled to room temperature, and then 8.95g (150 mmole) 2-ethanolamine was added. The resulting mixture was heatedto 80° C. for 20 hours, cooled to room temperature and diluted with 10mL water. The solution was dialyzed against water for 24 hours, thenlyophilized, yielding the polymer as a brittle white solid.

Example 5 Synthesis of poly(TMAEAC)

[0134] A solution of 48.25 g (0.25 mol) 2-trimethylammonioethylacrylatechloride in 400 mL isopropanol was degassed by nitrogen purging andheated to 35° C. To this stirred solution was added a solution of 0.8 gpotassium persulfate in 10 mL distilled water. A slight exotherm wasobserved. The solution was stirred at 35° C. for 6 hours, then cooled toroom temperature. The solution was added to hexanes and the resultingprecipitate was isolated by filtration.

Example 6 Synthesis ofpoly(decamethylenedimethylammonium-co-ethylenedimethylammonium) bromide

[0135] N,N,N′,N′-tetramethylethylenediamine (10.0 g, Aldrich),1,10-dibromodecane (25.8 g, Aldrich) and methanol (100 mL) were placedinto a three-neck 250 mL round bottom flask. The mixture was heated withgentle stirring to 65° C. for 6 days, at which point methanol (40 mL)was added, and the mixture was refluxed for an additional 2 days. Themixture was then dripped into acetone, forming a solid that wascollected by filtration, rinsed with acetone, and dried in a vacuum ovento yield 30.9 g of product.

Example 7 Synthesis of poly(TMAEMC-co-styrene) 75/25

[0136] A 500 mL round bottomed flask was charged withtrimethylammonioethyl-methacrylate chloride (26.0 g of a 70 wt % aqueoussolution, 18.2 g), styrene (6.0 g) and isopropanol (150 mL). Thesolution was degassed by the addition of a rapid stream of nitrogen for10 minutes, followed by the addition of AIBN (0.5 g). The solution wasdegassed for a further thirty minutes and, while continuing the additionof nitrogen, the solution was heated to 70° C., and the temperaturemaintained for 17 h. The polymer began to precipitate within 2 h, and bythe completion of the reaction a sticky white precipitate had formed.The reaction mixture was cooled, the isopropanol was decanted from thepolymer, and the polymer was dissolved in methanol. Dropwise addition ofthe methanol solution to ethyl acetate (1200 mL) caused the polymer toprecipitate as a fine white powder which was recovered by filtration.

Example 8 Synthesis of poly(TMAEMC-co-N-isopropylacrylamide) (67/33)

[0137] A 500 mL round bottomed flask was charged withtrimethylammonioethyl-methacrylate chloride (14.5 g of a 70 wt % aqueoussolution, 10.0 g), N-isopropylacrylamide (5.0 g) and isopropanol (150mL). The solution was degassed by the addition of a rapid stream ofnitrogen for 10 minutes, followed by the addition of AIBN (0.5 g). Thesolution was degassed for a further 60 minutes. The reaction mixture washeated to 70° C., and the temperature maintained for 16 h. The polymerpartially precipitated over the course of the reaction. Upon cooling,the propanol was decanted from the polymer, and the polymer wasdissolved in methanol. Precipitation of the methanol solution dropwiseinto ethyl acetate (1200 mL) caused the polymer to be deposited as whitecurds which were recovered by filtration, washed with ethyl acetate, anddried in vacuo.

[0138] Additional TMAEMC/N-isopropylacrylamide copolymers were preparedby a similar method with the starting monomers in the following ratios:

TMAEMC/N-isopropylacrylamide=40/60, 25/75 and 15/85.

Example 9 Synthesis of poly(MAPTAC-co-styrene) 75/25

[0139] To isopropanol (150 mL) was added a solution ofN-(3-trimethylammonio-propyl)methacrylamide chloride in water (50 wt %solution, 24.0 g, 12.0 g of monomer). To this solution was added styrene(6.0 g), followed by the addition of AIBN (0.5 g). The homogeneoussolution was degassed by bubbling a stream of nitrogen through it for 30minutes. The solution was heated to 70° C. for 15 h. The polymerpartially precipitated as the reaction proceeded. The solution wascooled, the isopropanol was decanted off, the white solid was washedwith propanol (50 mL). The propanol was decanted a second time, and thesolid was dissolved in methanol (150 mL). The clear solution was addeddropwise to ethyl acetate, causing the polymer to be precipitated as awhite powder. The polymer was recovered by filtration, washed with 50 mLof ethylacetate and air dried.

[0140] An additional MAPTAC/styrene copolymer was prepared by a similarmethod employing a 50/50 mixture of starting monomers.

Example 10 Synthesis of poly(TMAEMC-co-heptafluorobutylacrylate) 75/25

[0141] A 500 mL round bottomed flask was charged with2-trimethylammonioethyl-methacrylate chloride (26.0 g of a 70wt %aqueous solution, 18.2 g), heptafluorobutylacrylate (6.0 g) andisopropanol (150 mL). The solution was degassed by the addition of arapid stream of nitrogen for 10 minutes, followed by the addition ofAIBN (0.5 g). The solution was degassed for a further thirty minutesand, continuing the addition of nitrogen, the solution was heated to 70°C. The temperature was maintained for 17 h. The polymer began toprecipitate within 1 h, and by the completion of the reaction a stickywhite precipitate had formed. The reaction mixture was cooled, thepropanol was decanted from the polymer, and the polymer was dissolved inmethanol (100 mL). Precipitation of the methanol solution dropwise intoethyl acetate (1200 mL) caused the polymer to be deposited as a whitesolid which was recovered by filtration.

Example 11 Synthesis of poly(MAPTAC-co-N-t-butylacrylamide) 75/25

[0142] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 36.4 g of a 50%aqueous solution of N-(3-trimethylammonium-propyl)methacrylamidechloride and 6 g of N-t-butyl-acrylamide followed by 150 mL ofisopropanol. The solution was purged with nitrogen for 1 hour and 0.5 gAIBN was added. The mixture was purged for ˜15 minutes until all of theAIBN dissolved. The solution was heated to 75° C. under nitrogen for 16hours.

[0143] The resulting reaction mixture consisted of two phases. Theturbid liquid phase was decanted from the bulk of the reaction which wasa white sticky solid phase. The liquid was precipitated into 1200 mL ofethyl acetate and filtered by vacuum filtration through a Buchnerfunnel. The white hygroscopic precipitate was dried in vacuo. The solidphase was dissolved in methanol and precipitated into 1200 mL of ethylacetate and filtered by vacuum filtration to yield a white powder whichwas stored under vacuum.

[0144] Additional MAPTAC/N-t-butylacrylamide copolymers were prepared bya similar method beginning with the starting monomers in the followingratios: N-(3-trimethylammoniopropyl)methacrylamide/N-t-butyl-acrylamide=60/40, 50/50, 40/60, and 25/75.

Example 12 Synthesis ofpoly(N-decylallylamine-co-N-(4-trimethylammoniobutyl) allylamine)

[0145] To a solution of poly(allylamine).HCl (20.15 g of a 50 wt %aqueous solution) was added sodium hydroxide (5.64 g ) as a solid. Thesolution was stirred for 40 minutes, filtered and the filter cake waswashed with methanol (15 mL). The solution was further diluted withmethanol (25 mL) and to the solution was added 1-bromodecane (7.73 g, 35mmol) and (1-trimethylamino-4-bromobutane) chloride (9.13 g, 35 mmol). Asolution was prepared of sodium hydroxide (2.8 g, 70 mmol) in water (5mL). This solution was added to the reaction mixture in four portions atthirty minute intervals. The solution was then stirred at roomtemperature for 24 h, followed by dialysis against deionized water andfreeze-dried. A total of 23.2 g of a glassy, hygroscopic solid wasrecovered.

Example 13 Synthesis of poly(TMAEMC-co-N-t-butylacrylamide) 57/43

[0146] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 18.20 g of a 70%aqueous solution of 2-trimethylammonium-ethylmethacrylic chloride and9.7 g of N-t-butylacrylamide followed by 150 mL of isopropanol. Thesolution was purged with nitrogen for 1 hour and 0.5 g AIBN was added.The mixture was purged for ˜15 minutes until all of the AIBN dissolved.The solution was heated to 75° C. under nitrogen for 16 hours.

[0147] The resulting reaction mixture consisted of two easily separablephases. The liquid phase was decanted from the bulk of the reactionwhich was a white solid. The liquid was precipitated into 1200 mL ofethyl acetate and filtered by vacuum filtration through a Buchnerfunnel. The white precipitate was dried in vacuo and weighed: fractionA, 10.1 g (45.1% yield based on 22.4 g monomers added). The solid phasewas dissolved in methanol and precipitated into 600 mL of ethyl acetateand filtered by vacuum filtration to yield fraction B, 5.81 g of a whitepowder (25.9% yield) which was dried under vacuum.

[0148] TMAEMC/N-t-Butylacrylamide copolymers were also prepared by asimilar method with the starting monomers in the following ratios:

TMAEMC/N-t-Butylacrylamide=63/37, 50/50, 40/60, 25/75, 15/85 and 5/95.

Example 14 Synthesis of poly(MAPTAC-co-N-n-decylacrylamide) 75/25

[0149] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 36.4 g of a 50%aqueous solution of N-(3-trimethylammoniopropyl)methacrylamide chlorideand 6 g of N-n-decylacrylamide followed by 150 mL of isopropanol. Thesolution was purged with nitrogen for 1 hour and 0.5 g AIBN was added.The mixture was purged for 15 minutes until all of the AIBN dissolved.The solution was heated to 75° C. under nitrogen for 16 hours.

[0150] The reaction mixture consisted of two easily separable phases.The clear, yellow liquid phase was precipitated into 1200 mL of ethylacetate. The precipitate was isolated by filtration and dried undervacuum to yield 2.14 g of a yellow powder, fraction A (8.84% yield).Methanol was added to the creamy yellow reaction precipitate and theresulting turbid yellow solution was precipitated into 1200 mL of ethylacetate. The white precipitate was isolated by filtration and driedunder vacuum to yield fraction B, 17.22 g, as a slightly yellow powder(71.2% yield).

[0151] Additional MAPTAC/N-n-decylacrylamide copolymers were prepared bya similar method with the starting monomers in the following ratios:MAPTAC/N-n-decylacrylamide=60/40, 50/50, and 40/60.

Example 15 Synthesis of poly(TMAEMC-co-pentafluorostyrene) 75/25

[0152] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 26.0 g of a 70%aqueous solution of 2-trimethyl-ammonium-ethylmethacrylate chloride and6 g of pentafluorostyrene followed by 150 mL of isopropanol. Thesolution was purged with nitrogen for 1 hour and 0.5 g AIBN was added.The mixture was purged for ˜15 minutes until all of the AIBN dissolved.The solution was heated to 75° C. under nitrogen for 16 hours.

[0153] The reaction mixture consisted of two phases. The turbid solutionwas discarded. The bulk of the reaction, consisting of a white solidmass at the bottom of the flask, was dissolved in methanol. Theresulting clear solution was precipitated into 1200 mL of ethyl acetate.The white precipitate was isolated by vacuum filtration to yield 20.39 gof a fine white powder (84.3% yield).

[0154] Additional TMAEMC/pentafluorostyrene copolymers were prepared bya similar method with the starting monomers in the following ratios:

TMAEMC/pentafluorostyrene=60/40 and 50/50.

Example 16 Synthesis of poly(MAPTAC-co-pentafluorostyrene) 75/25

[0155] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 36.3 g of a 50%aqueous solution of N-(3-trimethylammoniopropyl)methacrylamide chlorideand 6 g of pentafluorostyrene followed by 150 mL of isopropanol. Thesolution was purged with nitrogen for 1 hour and 0.5 g AIBN was added.The mixture was purged for ˜15 minutes until all of the AIBN dissolved.The solution was heated to 75° C. under nitrogen for 16 hours.

[0156] The reaction mixture consisted of a turbid solution with a whiteprecipitate. The supernatant was discarded. The white reactionprecipitate was dissolved in methanol and the resulting clear solutionwas precipitated into 1200 mL of ethyl acetate. The white precipitatewas isolated by filtration and dried under vacuum to yield 12.81 g of afine white powder (52.9% yield).

[0157] Additional MAPTAC/pentafluorostyrene copolymers were prepared bya similar method with the starting monomers in the following ratios:

MAPTAC/pentafluorostyrene=60/40 and 50/50.

Example 17 Synthesis of MAPTAC/N-t-Butylacrylamide/HEMA Terpolymer33/33/33

[0158] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.1 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 8 g ofN-t-butylacrylamide, and 8 g of 2-hydroxyethylmethacrylate. The solutionwas purged with nitrogen for 1 hour and 0.5 g of AIBN was added. Themixture was purged for ˜15 min until all of the AIBN dissolved. Thesolution was heated to 75° C. under nitrogen for 16 hours.

[0159] The reaction mixture consisted of a turbid solution with a whitelatex in the bottom of the flask. The solution was precipitated into1200 mL of ethyl acetate. The white precipitate was isolated byfiltration to yield a sticky white powder which was dried under vacuumto yield 10.43 g of a lumpy white solid (fraction A) (43.1% yield). Thewhite reaction precipitate was dissolved in methanol and precipitatedinto 1200 mL of ethyl acetate. The precipitate was isolated byfiltration and dried under vacuum to yield 8.89 g of a fine white powder(fraction B) (36.7% yield).

[0160] Additional MAPTAC/N-t-butylacrylamide/HEMA terpolymers wereprepared by a similar method beginning with the following ratios of thestarting monomers:

MAPTAC/N-t-Butylacrylamide/HEMA=28/43/28, 23/53/23, and 18/63/18.

Example 18 Synthesis of MAPTAC/N-Isopropylacrylamide/HEMA Terpolymer18/63/18

[0161] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 8.9 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 15.3 g ofN-iso-propylacrylamide, and 4.4 g of 2-hydroxyethylmethacrylate. Thesolution was purged with nitrogen for 1 hour and 0.5 g of AIBN wasadded. The mixture was purged for 15 min until all of the AIBNdissolved. The solution was heated to 75° C. under nitrogen for 16hours.

[0162] The clear slightly pink reaction solution was precipitated into1200 mL of ethyl acetate. The precipitate was isolated by filtration toyield a sticky white solid which was dried under vacuum to yield 14.42 gof a hard clear/white granular solid (59.6% yield).

Example 19 Synthesis of MAPTAC/N-Decylacrylamide/HEMA Terpolymer33/33/33

[0163] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.1 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 8 g ofN-decylacrylamide, and 8 g of 2-hydroxyethylmethacrylate. The solutionwas purged with nitrogen for 1 hour and 0.5 g of AIBN was added. Themixture was purged for ˜15 min until all of the AIBN dissolved. Thesolution was heated to 75° C. under nitrogen for 16 hours.

[0164] The reaction mixture consisted of two phases. The clear yellowsolution was precipitated into 1200 mL of ethyl acetate. The precipitatewas isolated by filtration. The sticky yellow precipitate was driedunder vacuum and the resulting brittle clear yellow foam was crushed toyield 4.98 g of a fine yellow granular powder (fraction A) (20.6%yield). The white reaction latex was dissolved in methanol andprecipitated into 1200 mL of ethyl acetate. The precipitate was isolatedby filtration and dried under vacuum to yield 10.24 g of a slightlyyellow granular solid (fraction B) (42.3% yield).

[0165] Additional MAPTAC/N-Decylacrylamide/HEMA terpolymers wereprepared by a similar method beginning with the following ratios ofstarting monomers:

MAPTAC/N-Decylacrylamide/HEMA=28/43/28, 23/53/23, and 18/63/18.

Example 20 Synthesis of TMAEAC/n-Butylacrylate/Acrylamide Terpolymer10/30/60

[0166] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 7.26 g of n-butylacrylate, and14.52 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0167] The resulting white reaction mixture was filtered by vacuumfiltration through a Buchner funnel to yield a white powder. The powderwas washed with isopropanol and dried under vacuum to yield 21.57 g of afine white powder (89.1% yield based on 24.2 g of monomers).

[0168] Additional TMAEAC/n-butylacrylate/acrylamide terpolymers wereprepared by a similar method beginning with the following ratios ofstarting monomers:

TMAEMC/n-butylacrylate/acrylamide=20/20/60 and 30/10/60.

Example 21 Synthesis of TMAEAC/t-Butylacrylate/Acrylamide Terpolymer10/30/60

[0169] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 7.26 g of t-butylacrylate, and14.52 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0170] The resulting white reaction mixture was filtered by vacuumfiltration through a Buchner funnel to yield a white powder. The powderwas washed with isopropanol and dried under vacuum to yield 21.13 g of awhite powder (87.3% yield).

[0171] Additional TMAEAC/t-butylacrylate/acrylamide terpolymers wereprepared by a similar method beginning with the following ratios ofstarting monomers:

TMAEAC/t-butylacrylate/acrylamide=20/20/60 and 30/10/60.

Example 22 Synthesis of TMAEAC/n-Decylacrylate/Acrylamide Terpolymer10/30/60

[0172] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 7.26 g of n-decylacrylate, and14.52 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0173] The resulting white reaction mixture was filtered by vacuumfiltration through a Buchner funnel to yield a white powder. The powderwas washed with isopropanol and dried under vacuum to yield 21.52 g of afine white powder (89% yield).

[0174] Additional TMAEAC/n-decylacrylate/acrylamide terpolymers wereprepared by a similar method beginning with the following ratios ofstarting monomers:

TMAEAC/n-decylacrylate/acrylamide=20/20/60, and 30/10/60.

Example 23 Synthesis of MAPTAC/N-t-Butylmethacrylamide/MethacrylamideTerpolymer 10/30/60

[0175] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 7.26 g ofN-t-butylmethacrylamide, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g of AIBN was added. The mixturewas purged for ˜15 min until all of the AIBN dissolved. The solution washeated to 75° C. under nitrogen for 16 hours.

[0176] The white reaction mixture was too difficult to filter by vacuumfiltration so centrifugation techniques were employed instead. Thereaction mixture was poured into 50 mL centrifuge tubes and centrifuged.The supernatant was discarded. Isopropanol was added to the polymer andthe mixture was stirred and centrifuged. The supernatant was discardedand the white solids were combined and dried under vacuum to yield 14.99g of a slightly buff powder (61.9% yield).

[0177] Additional MAPTAC/N-t-butylmethacrylamide/methacrylamideterpolymers were prepared by a similar method beginning with thefollowing ratios of starting monomers:MAPTAC/N-t-butylmethacrylamide/methacrylamide=20/20/60, 33/33/33 and30/10/60.

Example 24 Synthesis of MAPTAC/n-Decylmethacrylate/MethacrylamideTerpolymer 10/30/60

[0178] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 7.26 g ofn-decylmethacrylate, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g of AIBN was added. The mixturewas purged for ˜15 min until all of the AIBN dissolved. The solution washeated to 75° C. under nitrogen for 16 hours.

[0179] The isopropanol was decanted leaving a white chunky powder.Isopropanol was added and the mixture was poured into 50 mL centrifugetubes and centrifuged. The supernatant was discarded. Isopropanol wasadded to the polymer and the mixture was stirred and centrifuged. Thesupernatant was discarded and the white solids were combined and driedunder vacuum to yield 18.50 g of a granular white solid (76.4% yield).

[0180] Additional MAPTAC/N-decylmethacrylamide/methacrylamideterpolymers were prepared by a similar method beginning with thefollowing ratios of starting monomers:MAPTAC/N-decylmethacrylamide/methacrylamide=20/20/60, 33/33/33 and30/10/60.

Example 25 Synthesis of TMAEMC/n-Decylmethacrylate/MethacrylamideTerpolymer 10/30/60

[0181] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 3.46 g of a 70% aqueous solution of2-trimethylammonioethylmethacrylate chloride, 7.26 g ofn-decylmethacrylate, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g AIBN was added. The mixturewas purged for ˜15 minutes until all of the AIBN dissolved. The solutionwas heated to 75° C. under nitrogen for 16 hours.

[0182] The white reaction mixture was poured into 50 mL centrifuge tubesand centrifuged. The supernatant was discarded. Isopropanol was added tothe polymer and the mixture was stirred and centrifuged. The supernatantwas discarded and the white solids were combined and dried under vacuumto yield 10.29 g of a hard white solid (42.5% yield).

[0183] Additional TMAEMC/N-n-decylmethacrylamide/methacrylamideterpolymers were prepared by a similar method beginning with thefollowing ratios of starting monomers:TMAEMC/N-n-decylmethacrylamide/methacrylamide=20/20/60, 33/33/33 and30/10/60.

Example 26 Synthesis of TMAEMC/N-t-Butylmethacrylamide/MethacrylamideTerpolymer 10/30/60

[0184] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 3.46 g of a 70% aqueous solution of2-trimethylammonioethylmethacrylate chloride, 7.26 g ofN-t-butylmethacrylamide, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g AIBN was added. The mixturewas purged for ˜15 minutes until all of the AIBN dissolved. The solutionwas heated to 75° C. under nitrogen for 16 hours.

[0185] The white reaction mixture was poured into 50 mL centrifuge tubesand centrifuged. The supernatant was discarded. Isopropanol was added tothe polymer and the mixture was stirred and centrifuged. The supernatantwas discarded and the white solids were combined and dried under vacuumto yield 18.35 g of a fine white powder (75.8% yield).

[0186] Additional TMAEMC/N-t-butylmethacrylamide/methacrylamideterpolymers were prepared by a similar method beginning with thefollowing ratios of starting monomers:TMAEMC/N-t-butylmethacrylamide/methacrylamide=20/20/60, 33/33/33 and30/10/60.

Example 27 Synthesis of TMAEMC/n-Butylmethacrylate/MethacrylamideTerpolymer 10/30/60

[0187] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 3.46 g of a 70% aqueous solution of2-trimethylammonioethylmethacrylate chloride, 7.26 g ofn-butylmethacrylate, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g AIBN was added. The mixturewas purged for ˜15 minutes until all of the AIBN dissolved. The solutionwas heated to 75° C. under nitrogen for 16 hours.

[0188] The white reaction mixture was poured into 50 mL centrifuge tubesand centrifuged. The supernatant was discarded. Isopropanol was added tothe polymer and the mixture was stirred and centrifiged. The supernatantwas discarded and the white solids were combined and dried under vacuumto yield 20.99 g of a clumpy white powder (86.7% yield).

[0189] Additional TMAEMC/N-n-butylmethacrylamide/methacrylamideterpolymers were prepared by a similar method beginning with thefollowing ratios of starting monomers:TMAEMC/N-n-butylmethacrylamide/methacrylamide=20/20/60 and 30/10/60.

Example 28 Synthesis of MAPTAC/n-Butylmethacrylate/MethacrylamideTerpolymer 10/30/60

[0190] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 4.84 g of a 50% aqueous solution ofN-(3-trimethylammoniopropyl)methacrylamide chloride, 7.26 g ofn-butylmethacrylate, and 14.52 g of methacrylamide. The solution waspurged with nitrogen for 1 hour and 0.5 g of AIBN was added. The mixturewas purged for ˜15 min until all of the AIBN dissolved. The solution washeated to 75° C. under nitrogen for 16 hours.

[0191] The white reaction mixture was filtered by vacuum filtration toyield a white powder. The powder was washed with isopropanol and driedunder vacuum to yield 22.20 g of a white powder (91.7% yield).

[0192] Additional MAPTAC/n-butylmethacrylate/methacrylamide terpolymerswere prepared by a similar method beginning with the following ratios ofstarting monomers:

MAPTAC/n-butylmethacrylate/methacrylamide=20/20/60 and 30/10/60.

Example 29 Synthesis of TMAEAC/n-Decylacrylamide/Acrylamide Terpolymer33/33/33

[0193] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of n-decylacrylamide,and 8.06 g of acrylamide. The solution was purged with nitrogen for 1hour and 0.5 g AIBN was added. The mixture was purged for ˜15 minutesuntil all of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0194] The reaction mixture was precipitated into 1200 mL of ethylacetate. The fine precipitate was filtered by vacuum filtration to yielda sticky yellow material. The light yellow solid was dissolved inmethanol and precipitated into 1200 mL of ethyl acetate. The precipitatewas filtered by vacuum filtration to yield 10.85 g of a sticky, slightlyyellow powder (44.8% yield).

Example 30 Synthesis of TMAEAC/N-t-Butylacrylamide/Acrylamide Terpolymer33/33/33

[0195] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of N-t-butylacrylamide,and 8.06 g of acrylamide. The solution was purged with nitrogen for 1hour and 0.5 g AIBN was added. The mixture was purged for ˜15 minutesuntil all of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0196] The reaction mixture consisted of a clear colorless solution witha small amount of white sticky solid. The clear solution wasprecipitated into 1200 mL of ethyl acetate. The white precipitate wasfiltered, dissolved in water, and lyophilized to yield 6.65 of a whitepowder (27.5% yield).

Example 31 Synthesis of TMAEAC/Styrene/Acrylamide Terpolymer 33/33/33

[0197] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of styrene, and 8.06 gof acrylamide. The solution was purged with nitrogen for 1 hour and 0.5g AIBN was added. The mixture was purged for ˜15 minutes until all ofthe AIBN dissolved. The solution was heated to 75° C. under nitrogen for16 hours.

[0198] The reaction mixture consisted of a clear colorless solution anda white solid. The clear solution was discarded. The solid was dissolvedin methanol, and precipitated into ethyl acetate (1200 mL). A whiteprecipitate formed which settled out of the solution as a sticky whitesolid. The ethyl acetate was decanted and the solid dried by passingnitrogen through the flask. The solid was dissolved in water andlyophilized to yield 18.14 g of a fine white powder (75% yield).

Example 32 Synthesis of TMAEAC/n-Butylacrylate/Acrylamide Terpolymer33/33/33

[0199] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of n-butylacrylate, and8.06 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0200] The reaction mixture consisted of a clear colorless solution anda white chunky solid. The solution phase was discarded and the whitesolid dissolved in water, filtered and lyophilized to yield 12.84 of afine white powder (53.1% yield).

Example 33 Synthesis of TMAEAC/n-Decylacrylate/Acrylamide Terpolymer33/33/33

[0201] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of n-decylacrylate, and8.06 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0202] The white reaction mixture was precipitated into 1200 mL of ethylacetate. The turbid solution was decanted and the polymer was dried withnitrogen, dissolved in water, and lyophilized to yield 8.79 g of finewhite powder (36.3% yield).

Example 34 Synthesis of TMAEAC/t-Butylacrylate/Acrylamide Terpolymer33/33/33

[0203] To a 500 mL round-bottom, three-neck flask fitted with athermocouple, reflux condenser, and septum was added 150 mL ofisopropanol followed by 16.13 g of a 50% aqueous solution of2-trimethylammonioethylacrylate chloride, 8.06 g of t-butylacrylate, and8.06 g of acrylamide. The solution was purged with nitrogen for 1 hourand 0.5 g AIBN was added. The mixture was purged for ˜15 minutes untilall of the AIBN dissolved. The solution was heated to 75° C. undernitrogen for 16 hours.

[0204] The white reaction mixture was precipitated into 1200 mL of ethylacetate. The turbid solution was decanted and the polymer was dried withnitrogen, dissolved in water, and lyophilized to yield 6.51 g of finewhite powder (26.9% yield).

Example 35 Synthesis of TMAEMC/n-hexyl methacrylate (75/25)

[0205] 2-Trimethylammonium ethyl methacrylic chloride (75 mol %, 1.875mol, 389.49 g, 556.42 g 70% aqueous solution) and n-hexyl methacrylate(25 mol %, 0.625 mol, 106.425 g) were dissolved in ethanol (3750 ml).The clear, pale yellow solution was degassed for 1.25 h. AIBN (3 mol %,75 mmol, 12.3 g) was added and the solution was degassed for anadditional 45 min. The polymerization was run at 70° C. for 16 h.

[0206] The polymer solution was precipitated into ethyl acetate (1:2).The polymer was redissolved in methanol (3500 ml) and reprecipitatedinto ethyl acetate (1:2). The sticky white polymer was washed with ethylacetate (3000 ml). The polymer became brittle and was left overnight todry in ethyl acetate (2000 ml). The resulting white brittle solid/powderwas filtered, crushed, and dried in vacuo (426.1 g).

Example 36 Synthesis of TMAEMC/n-hexyl methacrylate (60/40)

[0207] 2-Trimethylammonium ethyl methacrylic chloride (60 mol %, 1.5mol, 311.595 g, 445.14 g 70% aqueous solution) and n-hexyl methacrylate(40 mol %, 1 mol, 170.28 g) were dissolved in methanol (3750 ml). Theclear, pale yellow solution was degassed for 1.25 h. AIBN (3 mol %, 75mmol, 12.3 g) was added and the solution was degassed for an additional45 min. The polymerization was run at 65° C. for 16 h.

[0208] The solution was precipitated into ethyl acetate (1:6). Thepolymer was redissolved in ethanol and reprecipitated into ethyl acetate(1:3). The polymer was washed twice with ethyl acetate (2000 ml total)to give a fine white precipitate.

[0209] The precipitate was dried in vacuo to yield a white powder(284.66 g).

Example 37 Synthesis of TMAEMC/styrene (55/45)

[0210] 2-Trimethylammonium ethyl methacrylic chloride (55 mol %, 1.375mol, 285.63 g, 408.04 g 70% aqueous solution) and styrene (45 mol %,1.125 mol, 117.17 g, 128.9 ml) were dissolved in methanol (2500 ml). Theclear, pale yellow solution was degassed for 1.25 h. AIBN (2 mol %, 50mmol, 8.2 g) was added and the solution was degassed for an additional45 min. The polymerization was run at 65° C. for 16 h. The solution wasprecipitated into ethyl acetate (1:6). The white polymer was washedtwice with ethyl acetate (2000 ml total). The powder was redissolved inethanol (1750 ml) and reprecipitated into ethyl acetate (1:3). Thepolymer was washed twice with ethyl acetate (2000 ml total) andfiltered. The precipitate was dried in vacuo to yield a white powder(387.51 g).

Example 38 Reaction of Poly(vinylamine) with 10 mol % n-hexyl bromide

[0211] Poly(vinylamine) (3.48 mol, 150 g, 461.53 g 32.5% aqueoussolution, Mw 23K) was dissolved in ethanol (900 ml) followed by theaddition of n-hexyl bromide (10 mol %, 0.348 mol, 49 ml). The resultingclear yellow solution was heated to 70° C., and sodium hydroxide (32 gof a 50 wt % solution) was added in 4 equal portions at 1 hourintervals. Heating was continued for 16 h.

[0212] The clear orange reaction solution was precipitated intoisopropanol acidified with 10% hydrochloric acid (4000 mlisopropanol/400 ml hydrochloric acid). The orange polymer was washedwith isopropanol (1500 ml) and broken into small pieces. The polymer wasredissolved in water (1250 ml) and poured into isopropanol (1:3). Theresulting solution was milky with no precipitate. More hydrochloric acidwas added upon which fine white polymer precipitated. Hydrochloric acidwas added until no further precipitate formed (122 ml). Isopropanol wasadded (2500 ml) to the precipitate/acidic isopropanol mixture and themixture was allowed to stand overnight.

[0213] The precipitate was filtered and dried in vacuo (296.47 g).

Example 39 Reaction of Poly(ethyleneimine) with 20 mol % n-hexyl bromide

[0214] Poly(ethyleneimine) (8.12 mol, 350 g, 700 g 50% aqueous solution)was dissolved in ethanol (2100 ml) followed by n-hexyl bromide (1.62mol, 268.2 g, 228.1 ml) to give a clear yellow solution. The solutionwas heated to 70° C., and sodium hydroxide (136 mL of a 50 wt %solution) was added in 4 equal portions at 1 hour intervals. Heating wascontinued for 16 h.

[0215] The slightly turbid yellow solution was precipitated intoisopropanol acidified with 10% hydrochloric acid (1:4). The stickyprecipitate was allowed to sit in ethyl acetate overnight. The ethylacetate was decanted and the sticky yellow polymer was dissolved in aminimum amount of water and reprecipitated into isopropanol (1:4). Theprecipitate was washed with ethanol and allowed to dry in vacuo. Theresulting brittle polymer was crushed to yield a yellow powder (531.5g).

Example 40 Reaction of Poly(ethyleneimine) with 10 mol % (4-bromobutyl)trimethylammonium bromide and 20 mol % 1-bromo-3-phenylpropane

[0216] (4-bromobutyl)trimethylammonium bromide was prepared by thereaction of trimethylamine and 1,4-dibromobutane in methanol.

[0217] Poly(ethyleneimine) (8.12 mol, 350 g, 700 g of a 50% aqueoussolution) was dissolved in ethanol (2100 ml) followed by(4-bromobutyl)trimethylammonium bromide (0.812 mol, 223.5 g) and1-bromo-3-phenylpropane (1.63 mol, 247 ml). The slightly turbid yellowsolution was heated to 70° C., and sodium hydroxide (208 g of a 50 wt %solution) was added in 4 equal portions at 1 hour intervals. Thesolution was heated for a total of 16 h. for 16 h.

[0218] The turbid dark yellow reaction solution was precipitated intoisopropanol acidified with 10% hydrochloric acid (1:3). The stickyyellow precipitate was washed twice with isopropanol (1000 ml),redissolved in water (2000 ml), and reprecipitated into isopropanol(1:3). The sticky polymer was washed several times with isopropanol anddried in vacuo. The resulting brittle yellow polymer was crushed toyield a yellow granular powder (802.9 g).

Example 41 Reaction of Poly(allylamine) with 10 mol %(4-bromobutyl)trimethyl-ammonium bromide and 10 mol % n-hexyl bromide

[0219] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g of a 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 75° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml) and(4-bromobutyl)trimethylammonium bromide (0.428 mol, 117.63 g). Sodiumhydroxide (0.428 mol, 17.12 g, 34.26 g 50% aqueous solution) was addedin 4 equal portions over 4 h. The reaction was allowed to proceed for atotal of 16 h.

[0220] Bromohexane (0.428 mol, 70.65 g) was added to the reactionmixture at 75° C. Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g 50%aqueous solution) was added in 4 equal portions over 4 h and thereaction was allowed to proceed for a total of 16 h.

[0221] The reaction solution was precipitated into isopropanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed twicewith isopropanol (3000 ml) and filtered.

[0222] The polymer was redissolved in water (500 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed twice withisopropanol (4000 ml) and filtered. The polymer was dried in vacuo toyield a powder (600 g).

Example 42 Reaction of Poly(allylamine) with 10 mol % (3-chloropropyl)dimethyloctylammonium bromide

[0223] (3-chloropropyl)dimethyloctylammonium bromide was prepared by thereaction of 1-bromo-3-chloropropane and dimethyloctylamine in methanol.

[0224] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g of a 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 70° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml),(3-chloropropyl)dimethyl-octylammonium bromide (0.428 mol, 134.61 g),and water (300 ml). Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g 50%aqueous solution) was added in 4 equal portions over 4 h. The reactionwas allowed to proceed for a total of 16 h.

[0225] The reaction solution was precipitated into ethanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed withisopropanol (3000 ml) and filtered.

[0226] The polymer was redissolved in water (1000 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed with isopropanol(4000 ml) and filtered. The polymer was dried in vacuo to yield a powder(600 g).

Example 43 Reaction of Poly(allylamine) with 10 mol % (3-chloropropyl)dimethyloctylammonium bromide and 10 mol % benzyl bromide

[0227] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g of a 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 70° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml),(3-chloropropyl)dimethyl-octylammonium bromide (0.428 mol, 134.61 g),and water (300 ml). Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g 50%aqueous solution) was added in 4 equal portions over 4 h. The reactionwas allowed to proceed for a total of 17 h.

[0228] Benzyl bromide (0.428 mol, 73.21 g, 50.91 ml) was added to thereaction mixture at 70° C. Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g50% aqueous solution) was added in 4 equal portions over 4 h and thereaction was allowed to proceed for a total of 16 h.

[0229] The reaction solution was precipitated into isopropanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed withisopropanol and filtered

[0230] The polymer was redissolved in water (500 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed twice withisopropanol (4000 ml) and filtered. The polymer was dried in vacuo toyield a powder (600 g).

Example 44 Reaction of Poly(allylamine) with 10 mol % n-hexyl bromide

[0231] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g of a 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 75° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml) and n-hexyl bromide(0.428 mol, 70.65 g). Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g 50%aqueous solution) was added in 4 equal portions over 4 h. The reactionwas allowed to proceed for a total of 16 h.

[0232] The reaction solution was precipitated into isopropanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed twicewith isopropanol (3000 ml) and filtered.

[0233] The polymer was redissolved in water (500 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed twice withisopropanol (4000 ml) and filtered. The polymer was dried in vacuo toyield a powder (600 g).

Example 45 Reaction of Poly(allylamine) with 10 mol %(bromomethyl)cyclohexane

[0234] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 75° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml) and(bromomethyl)cyclohexane (0.428 mol, 75.79 g). Sodium hydroxide (0.428mol, 17.12 g, 34.26 g 50% aqueous solution) was added in 4 equalportions over 4 h. The reaction was allowed to proceed for a total of 16h.

[0235] The reaction solution was precipitated into isopropanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed twicewith isopropanol (3000 ml) and filtered.

[0236] The polymer was redissolved in water (500 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed twice withisopropanol (4000 ml) and filtered. The polymer was dried in vacuo toyield a powder (500 g).

Example 46 Reaction of Poly(allylamine) with 10 mol %(3-bromopropyl)trimethyl-ammonium bromide and 10 mol % benzyl bromide

[0237] Poly(allylamine) hydrochloride (4.28 mol, 400 g, 800 g 50%aqueous solution) was dissolved in ethanol (850 ml) and heated to 75° C.Sodium hydroxide (66 mol %, 2.81 mol, 112.98 g, 225.98 g 50% aqueoussolution) was added followed by water (500 ml) and(3-chloropropyl)dimethyloctylammonium bromide (0.428 mol, 111.63 g) andwater (300 ml). Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g 50%aqueous solution) was added in 4 equal portions over 4 h. The reactionwas allowed to proceed for a total of 16 h.

[0238] Benzyl bromide (0.428 mol, 73.21 g, 50.91 ml) was added to thereaction mixture at 70° C. Sodium hydroxide (0.428 mol, 17.12 g, 34.26 g50% aqueous solution) was added in 4 equal portions over 4 h and thereaction was allowed to proceed for a total of 16 h.

[0239] The reaction solution was precipitated into isopropanol acidifiedwith 20% hydrochloric acid (5000 ml). The precipitate was washed withisopropanol and filtered.

[0240] The polymer was redissolved in water (600 ml) and reprecipitatedinto isopropanol (4000 ml). The precipitate was washed twice withisopropanol (4000 ml) and filtered. The polymer was dried in vacuo toyield a powder (500 g).

Example 47 Reaction of Poly(ethyleneimine) with 10 mol %1-(3-chloropropyl)pyridinium bromide

[0241] 1-(3-chloropropyl)pyridinium bromide was prepared by the reactionof pyridine and 1-bromo-2-chloropropane. Pyridine (66 mL, 64.35 grams,0.81 moles), 1,3-dibromopropane (166.23 grams, 0.82 moles) andtetrahydrofuran (150 mL) were added to a 1 L, round bottom flaskequipped with air condensers and a magnetic stirring plate. The reagentswere allowed to react at room temperature for 4 days. The reactionbecame cloudy as precipitate accumulated. Solids were collected byvacuum filtration, resuspended in tetrahydrofuran (250 mL) and collectedby vacuum filtration. Solids were dried under vacuum at 35° C. for 24hours. Yield 63.64 grams (0.27 moles, 30%).

[0242] Poly(ethyleneimine) (0.67 mol, 30 g, 60 g of a 50% aqueoussolution) was diluted with water (160 ml). To this solution was added1-(3-chloropropyl)pyridinium bromide (15.84 g, 67 mmol). The solutionwas heated to 65 oC. Sodium hydroxide (67 mmol, 5.36 g of a 50 wt %solution) was added in four equal portions, spaced one hour apart. Thesolution was heated for a further 12 hours after the last addition ofsodium hydroxide (for a total heating time of 16 hours). The slightlycloudy yellow solution was cooled and precipitated into a solution of12M hydrochloric acid (75 ml) in isopropanol (1 L). The polymer wasrecovered by filtration, redissolved in water (300 mL) and precipitatedinto isopropanol. The polymer was recovered by filtration and dried at40° C. in vacuo.

Example 48 Reaction of Poly(vinylamine) with 10 mol % 1-(3-chloropropyl)pyridinium bromide

[0243] Poly(vinylamine) (227 mmol, 10 g, 30 g of a 32.5wt % aqueoussolution) was diluted with water (150 ml). To this solution was added1-(3-chloropropyl)pyridinium bromide (5.37 g, 22.7 mmol). The solutionwas heated to 75° C. Sodium hydroxide (22.7 mmol, 1.8 g of a 50 wt %solution) was added in three equal portions, spaced one hour apart. Thesolution was heated for a further 21 hours after the last addition ofsodium hydroxide (for a total heating time of 24 hours). The clearsolution was cooled and precipitated into a solution of 5% conc.hydrochloric acid in methanol (1200 ml). The very fine white polymer wasrecovered by filtration, washed with methanol, briefly air dried anddried in vacuo for 36 hours.

Example 49 Reaction of Poly(ethyleneimine) with 20 mol % decyl bromideand 10 mol % (4-bromobutyl)trimethylammonium bromide

[0244] A solution was prepared of poly(ethyleneimine) (50 g of a 50wt %aquous solution, 0.58 mol) in water (400 ml). To this solution was added(4-bromobutyl) trimethylammonium bromide (15.9 g, 58 mmol) in oneportion. The solution was heated to 65° C., and to the clear yellowsolution was added a solution of sodium hydroxide (4.64 g of a 50wt %solution, 58 mmol) in three equal portions, spaced one hour apart. Thesolution was heated for a total of 12 hours, after which time decylbromide (25.6 g, 116 mmol) was added in one portion. A further 9.28 g ofa 50 wt % solution of sodium hydroxide was added in three portions,spaced one hour apart, and the solution was heated for a final period of16 hours. The solution was cooled and precipitated in a solution of 5%conc. hydrochloric acid in methanol (2.5 L). The white polymer wasfiltered, washed with methanol (200 ml), redissolved in water (500 ml)and precipitated into iso-propanol (1200 ml). The product was recoveredby filtration, washed with propanol and dried in vacuo. Yield 86%.

Example 50 Reaction of Poly(ethyleneimine) with 20 mol % n-hexylbromideand 10 mol % (3-bromopropyl)trimethylammonium bromide

[0245] A solution was prepared of poly(ethyleneimine) (50 g of a 50w %aqueous solution, 0.58 mol) in water (375 ml). To this solution wasadded (3-bromopropyl) trimethylammonium bromide(15.1 g, 58 mmol) in oneportion. The solution was heated to 65° C., and to the clear yellowsolution was added a solution of sodium hydroxide (4.64 g of a 50 wt %solution, 58 mmol) in three equal portions, spaced one hour apart. Thesolution was heated for a total of 10 hours, after which time n-hexylbromide (19.14 g, 116 mmol) was added in one portion. A further 9.28 gof a 50 wt % solution of sodium hydroxide was added in three portions,spaced one hour apart, and the solution was heated for a final period of14 hours. The solution was cooled and precipitated in a solution of 5%hydrochloric acid in methanol (2.3 L). The white polymer was filtered,washed with methanol (200 ml), redissolved in water (500 ml) andprecipitated into isopropanol (1200 ml). The product was recovered byfiltration, washed with propanol and dried in vacuo. Yield 81%.

Example 51 Reaction of Poly(allylamine) with 10 mol %1-(3-chloropropyl)pyridinium bromide

[0246] Poly(allylamine) hydrochloride (428 mmol, 40 g, 80 g 50% aqueoussolution) was dissolved in water (200 ml) and heated to 70° C. Sodiumhydroxide (66 mol %, 0281 mmol, 11.2 g, 22.4 g of 50% aqueous solution)was added. To this solution was added 1-(3-chloropropyl)pyridiniumbromide (10.1 g, 42.8 mmol dissolved in 50 ml of water). Sodiumhydroxide (42.8 mmol, 1.7 g, 3.4 g of 50% aqueous solution) was added in3 equal portions over 4 h. The reaction was allowed to proceed for atotal of 16 h. The reaction solution was precipitated into ethanolacidified with 10% hydrochloric acid (2000 ml). The precipitate waswashed with isopropanol (300 ml) and filtered. The polymer wasredissolved in water (200 ml), reprecipitated into isopropanol (800 ml)and dried in vacuo.

Example 52 Preparation of 3% Poly(allylamine/epichlorohydrin)

[0247] To a 4-L plastic beaker was added poly(allylamine) hydrochloride(2001.5 g of 50% aqueous solution; Nitto Boseki PAA-HCl-3L) and water(3L). The mixture was stirred until homogeneous and the pH was adjustedto ˜10.5 with solid NaOH (280.3 g). The pH was reduced by addingconcentrated hydrochloric acid until the pH was ˜10.2. The solution wasallowed to cool to room temperature in the beaker and epichlorohydrin(25 mL; 29.1 g, 3 mole %) was added all at once with stirring. Themixture was stirred gently until it gelled and then was allowed tocontinue curing for 18 h at room temperature. The gel was then removedand broken up by passing it through a Kitchen Aid mixer. The solid wasthen suspended in ˜16 L of deionized water. The gel was collected byfiltration and washed on the funnel until the conductivity of theeffluent was equal to 16.7 mS/cm. The solid was dried in a forced airoven at 60° C. for 5 days to yield 866.3 g of a granular, brittle, whitesolid. The solid was ground in a coffee grinder and passed through a 30mesh sieve.

Example 53 Reaction of Poly(ethyleneimine) with 10 mol % benzyl bromide

[0248] Poly(ethyleneimine) (8.12 mol, 350 g, 700 g 50% aqueous solution)was dissolved in ethanol (2100 ml), followed by the addition of benzylbromide (0.81 mol, 138.5 g). The solution was heated at 70° C. and tothis solution was added sodium hydroxide (32.4 g, 64.8 g of a 50wt %solution) in four portions spaced one hour apart. The solution washeated for a further 16 hours. The slightly turbid yellow solution wasprecipitated into isopropanol acidified with 10% hydrochloric acid (1:4)(5000 ml). The polymer was recovered by filtration, redissolved in water(1000 ml) and reprecipitated into propanol (3000 ml). The polymer wasrecovered by filtration and dried in vacuo.

Example 54 Reaction of Poly(allylamine) with 10 mol % n-decyl bromideand 10 mol % (10-bromodecyl)trimethylammonium bromide

[0249] Poly(allylamine) hydrochloride (428 mmol, 40 g, 80 g of a 50%aqueous solution) was dissolved in water (200 ml) and heated to 70° C.Sodium hydroxide (66 mol %, 281 mmol, 11.2 g, 22.4 g of 50% aqueoussolution) was added. To this solution was added(10-bromodecyl)trimethylammonium bromide (15.3 g, 42.8 mmol dissolved in50 ml of water). Sodium hydroxide (42.8 mmol, 1.7 g, 3.4 g of 50%aqueous solution) was added in 3 equal portions over 4 h. The reactionmixture was heated for a further 16 h. Decyl bromide (9.45 g, 42.8 mmol)was added in one portion, followed by the addition of sodium hydroxide(42.8 mmol, 1.7 g, 3.4 g of 50% aqueous solution) which was added in 3equal portions over 4 h. The reaction was heated for a further 12 hours,cooled and precipitated into 1000 ml of ethanol containing 50 ml ofconc. hydrochloric acid. The polymer was recovered by filtration andwashed with ethanol (200 ml).

Example 55 Preparation of 4.5% cross-linked poly(diallylmethylamine)

[0250] 83 g of an aqueous solution of poly(diallylmethylaminehydrochloride) (PAS-M-1, Lot No. 51017; Nitto Boseki Co.) was dilutedwith 170 mL deionized water. While stirring, 6.8 g NaOH was added to thepolymer solution. The reaction mixture was allowed to stir until allNaOH had dissolved. When the temperature of the solution had dropped tobelow 30° C., epichlorohydrin (1.2 mL) was added and stirring continued.The reaction medium slowly became viscous and after about 80 minutes,had gelled and the stirring was stopped. The polymer gel was left atroom temperature for an additional 60 hr. The polymer slab was brokeninto smaller pieces and dispersed in 400 mL deionized water. Theresulting suspension was stirred for 2 hr and then filtered. The swollenpolymer particles were resuspended in 600 mL deionized water, stirredfor 45 minutes and collected by filtration. The process was repeatedwith 800 mL water and 1 hr stirring. After filtration, the filtrateshowed a conductivity of 4 mS/cm. The filtered polymer (swollen gel) wasdried in a forced air oven at 60° C. to yield 42 g of product.

Example 56 Alkylation of Crosslinked Poly(diallylmethylamine) with1-bromodecane

[0251] 10 g of the ground polymer (Example 54) taken in a 1 liter3-necked round bottom flask was suspended in 150 mL deionized water. Thepolymer swelled significantly and was stirred with a mechanical stirrer.While stirring, 2 g 50% aqueous NaOH solution was added and thesuspension was stirred for 15 minutes. To the suspension was then addedwas added 12.5 g 1-bromodecane dissolved in 32 mL ethanol and thereaction mixture was stirred for 2 hours. 1 g of 50% aqueous sodiumhydroxide was then added and the reaction mixture was stirred at roomtemperature for 40 minutes followed by heating to 75° C. for 2 hr. 2 gNaOH solution was then added. The reaction mixture was stirred at 75° C.for an additional 18 hours, after which time heating was discontinued.After cooling to 30° C., 2 mL concentrated HCl was added and stirringwas continued for 15 minutes. The polymer was filtered and washed with200 mL deionized water, stirred with 200 mL water for fifteen minutesand filtered. This process was repeated twice and the filtered polymerwas suspended in 400 mL 2M NaCl solution, stirred for 45 minutes andfiltered. After removing the solvent by filtration, the polymer wassuspended in 500 mL of 2 M NaCl solution and stirred for 40 minutes. Thepolymer was filtered and this process of NaCl treatment was repeated twomore times. The filtered polymer was suspended in 400 mL deionizedwater. After stirring for 30 minutes the polymer was filtered andresuspended in 400 mL deionized water and stirred for 40 minutes.Concentrated HCl (1 mL) was added to the suspension and the mixture wasstirred for 20 minutes. The pH of the suspension was found to be 2.25.After stirring for an additional 20 minutes, the polymer was filteredand dried at 60° C. in a forced air oven, yielding 16.8 g of thealkylated polymer. The polymer was ground and passed through a 140 meshsieve.

Example 57 Reaction of 6%-cross-linked poly(allylamine) with 140 mol %6-bromo-hexane and 170 mol % (6-bromohexyl)trimethylammonium bromide

[0252] Methanol (5 L) and sodium hydroxide (133.7 g) were added to a 12L round bottom flask equipped with a mechanical stirrer, a thermometerand a condenser. After the solid dissolved, 297 g 6%epichlorohydrin-cross-linked polyallylamine was added along withadditional methanol (3L). (6-Bromohexyl)trimethylammonium bromide (522.1g) and 1-bromodecane (311.7 g) were added and the mixture was heated to65° C. with stirring. After 18 hours at 65° C., the mixture was allowedto cool to room temperature. The solid was filtered off and rinsed bysuspending, stirring for 30 minutes and filtering off the solid from1.2×12 L methanol, 2.2×22 L aqueous NaCl (2 M), 3.3×22 L deionizedwater, 4.1×22 L isopropanol. The resulting solid was dried in a vacuumoven at 50° C. to yield 505.1 g of an off-white solid. The solid wasthen ground to pass through an 80 mesh sieve.

Example 58 Reaction of poly(allylamine) with 8 mole percentepichlorohydrin

[0253] To a 5 gallon bucket was added poly(allylamine) hydrochloride(2.5 kg) and water (10 L). The mixture was stirred until homogeneous andthe pH was adjusted to 10 with solid NaOH. The solution was allowed tocool to room temperature in the bucket and epichlorohydrin (250 mL) wasadded all at once with stirring. The mixture was stirred gently until itgelled and then was allowed to continue curing for 18 h at roomtemperature. The gel was then removed and put into a blender withisopropanol (about 7.5 L). The gel was mixed in the blender with about500 mL isopropanol for about 3 minutes to form coarse particles and thesolid was collected by filtration. The solid was rinsed three times bysuspending it in 9 gallons of water, stirring the mixture for one hourand collecting the solid by filtration. The solid was rinsed once bysuspending it in isopropanol (60 L) stirring the mixture for one hourand collecting the solid by filtration. The solid was dried in a vacuumoven for 18 hours to yield 1.55 kg of a granular brittle white solid.

Example 59 Shiga Toxin binding assay

[0254] The ability of several polymers to bind Shiga type 1 and Shigatype 2 toxins was assessed by an EIA analysis. Stock solutions of eachpolymer were prepared by dissolving 100 mg of polymer in 1 ml ofdeionized water. To each solution was added 25 μl of a Shiga toxin stocksolution comprising either type 1 Shiga toxin or type 2 Shiga toxin (1μg/ml). The polymer-toxin solutions were incubated for 5.5 hours and 100μl of each solution was then analyzed by an EIA analysis. The EIAanalysis is a spectrophotometric analysis where the decrease offluorescence detected at 450 nm (relative to control) is a measure ofthe efficiency of toxin binding. A fluorescence intensity of <0.3absorbance units, that is, less than about 10% of the control intensity,is considered to be indicative of highly effective toxin binding.Fluorescence intensity values between about 0.30 and about 1.8 areconsidered indicative of moderate, but still significant, levels oftoxin binding.

[0255] As set forth in the Table below, each of the tested polymersexhibited at least moderate binding of each Shiga toxin. Each of thepolymers is identified by the number of the example which describes itssynthesis. Six of the eight polymers showed highly effective binding oftype 1 Shiga toxin, while four of the eight polymers were highlyeffective against type 2 Shiga toxin. TABLE 1 Fluorescence FluorescenceIntensity Intensity Polymer Type 1 Toxin Type 2 Toxin Control 3.0002.989 Example 48 0.275 0.478 Example47 0.220 0.113 Example 49 0.1060.130 Example 50 0.137 0.066 Example 51 1.606 1.726 Example 53 0.5980.790 Example 39 0.092 0.081 Example 51 0.151 1.422

Example 60 Clostridium difficile toxin binding assay

[0256] Polymers were diluted in 0.9% saline to a working concentrationof 1 mg/mL. 225 mL of this solution was then mixed with 25 mL of a 1:10dilution of culture supernatant from a clinical isolate of C. difficilewhich is known to produce both toxins A and B. A positive controlconsisting of 0.9% saline was set up in each assay. The mixture was thenincubated at room temperature on a rotator for approximately 60 minutes.The mixture was then centrifuged and the supernatants were then assayeddirectly for C. difficile toxins using the Cytoclone A+B enzymeimmunoassay (Meridian Diagnostics Inc.). The protocol provided by themanufacturer was used. This method employs an enzyme immunoassayspectrophotometric analysis where the decrease in fluorescence at 450 nm(relative to control) is a measure of the extent of toxin binding.

[0257] The results of this assay for a series of polymers is shown inTable 2. TABLE 2 Polymer Fluorescence Intensity Control 0.743Cholestipol 0.744 Cholestyramine 0.717 Example 58 0.24 Example 57 0.236Example 56 0.453 Example 52 0.134

[0258] Colestipol and Cholestyramine are commercially available ionexchange resins marketed for the reduction of bile acids. Colestipol isa copolymer of diethylenetriamine and epichlorohydrin. Cholestyramine isan ammonio-substituted styrene/divinylbenzene resin.

[0259] While this invention has been particularly shown and describedwith references to preferred embodiments thereof, it will be understoodby those skilled in the art that various changes in form and details maybe made therein without departing from the scope of the inventionencompassed by the appended claims.

What is claimed is:
 1. A method for inhibiting a pathogenic toxin in amammal, comprising the step of administering to the mammal atherapeutically effective amount of a polymer characterized by a repeatunit of comprising a plurality of Formula I,

wherein R is a hydrogen atom or a methyl or ethyl group; X is a covalentbond, a carbonyl group or a CH₂ group; Y is an oxygen atom or an NH orCH₂ group; Z is an aliphatic spacer group; R¹, R² and R³ are each,independently, a hydrogen atom, a normal or branched, substituted orunsubstituted C₁-C₂₄-alkyl group, aryl or arylalkyl group; and m and nare each, independently, 1 or 0; or —N⁺(R¹)(R²)(R³) is a heteroarylgroup; and A⁻ is a pharmaceutically acceptable anion; or a free basethereof.
 2. The method of claim 1 wherein Z is a normal or branchedC₂-C₂₄-alkylene group or a C₂-C₂₄-alkylene group interrupted at one ormore points by a heteroatom.
 3. The method of claim 2 wherein theheteroatom is a nitrogen, oxygen or sulfur atom.
 4. The method of claim3 wherein at least one of R¹, R² and R³ is an aryl group, a benzyl groupor a normal or branched, substituted or unsubstituted C₁-C₂₄-alkylgroup.
 5. The method of claim 3 wherein R¹, R² and R³ are each hydrogen.6. The method of claim 1 wherein the polymer is further characterized bya difunctional cross-linking monomer.
 7. The method of claim 6 whereinthe difunctional cross-linking monomer is selected from the groupconsisting of diacrylates, triacrylates and tetraacrylates,dimethacrylates, diacrylamides, diallylacrylamide, di(methacrylamides),triallylamine and tetraaleylammonium ion.
 8. The method of claim 7wherein the difunctional cross-linking monomer is selected from thegroup consisting of ethylene glycol diacrylate, propylene glycoldiacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate,butylene glycol dimethacrylate, methylene bis(methacrylamide), ethylenebis(acrylamide), ethylene bis(methacrylamide), ethylidenebis(acrylamide), ethylidene bis(methacrylamide), pentaerythritoltetraacrylate, trimethylolpropane triacrylate, bisphenol Adimethacrylate, bisphenol A diacrylate and divinylbenzene.
 9. The methodof claim 1 wherein the polymer is crosslinked by a bridging unitselected from the group consisting of straight chain or branched,substituted or unsubstituted alkylene groups, diacylalkylene groups,diacylarene groups and alkylene bis(carbamoyl) groups.
 10. The method ofclaim 9 wherein the bridging units are selected from the groupconsisting of —(CH₂)_(n)—, wherein n is an integer from about 2 to about20; —CH₂—CH(OH)—CH₂—;—C(O)CH₂CH₂C(O)—; —CH₂—CH(OH)—O—(CH₂)_(m)—O—CH(OH)—CH₂—, wherein m is 2 to about 4; —C(O)—(C₆H₂(COOH)₂)—C(O)—; and—C(O)NH(CH₂)_(p)NHC(O)—, wherein p is an integer from about 2 to about20.
 11. A method for inhibiting a pathogenic toxin in a mammal,comprising the step of administering to the mammal a therapeuticallyeffective amount of a polymer characterized by a repeat unit of theformula

wherein R¹, R² and R³ are each hydrogen.
 12. The method of claim 11wherein the polymer is crosslinked by a bridging unit selected from thegroup consisting of straight chain or branched, substituted orunsubstituted alkylene groups, diacylalkylene groups, diacylarene groupsand alkylene bis(carbamoyl) groups.
 13. The method of claim 12 whereinthe bridging units are selected from the group consisting of—(CH₂)_(n)—, wherein n is an integer from about 2 to about 20;—CH₂—CH(OH)—CH₂—; —C(O)CH₂CH₂C(O)—; —CH₂—CH(OH)—O—(CH₂)_(m)—O—CH(OH)—CH₂—, wherein m is 2 to about 4; —C(O)—(C₆H₂(COOH)₂)—C(O)—; and—C(O)NH(CH₂)_(p)NHC(O)—, wherein p is an integer from about 2 to about20.
 14. The method of claim 13 wherein the bridging units are—CH₂—CH(OH)—CH₂—.
 15. A method for inhibiting a pathogenic toxin in amammal, comprising the step of administering to the mammal atherapeutically effective amount of a polymer characterized by a firstrepeat unit of Formula III,

wherein both R¹ and R² are hydrogen; and a second repeat unit of FormulaIII wherein R¹ and R² are each, independently, a C₁-C₂₄-alkyl group. 16.The method of claim 15 wherein in the second repeat unit of Formula III,R¹ is a methyl group and R² is a linear or branched C₁-C₁₈ alkyl group.17. The method of claim 15 wherein the polymer is cross-linked.
 18. Themethod of claim 11 wherein the polymer is crosslinked by a bridging unitselected from the group consisting of straight chain or branched,substituted or unsubstituted alkylene groups, diacylalkylene groups,diacylarene groups and alkylene bis(carbamoyl) groups.
 19. The method ofclaim 12 wherein the bridging units are selected from the groupconsisting of —(CH₂)_(n)—, wherein n is an integer from about 2 to about20; —CH₂—CH(OH)—CH₂—; —C(O)CH₂CH₂C(O)—; —CH₂—CH(OH)—O—(CH₂)_(m)—O—CH(OH)—CH₂—, wherein m is 2 to about 4; —C(O)—(C₆H₂(COOH)₂)—C(O)—; and—C(O)NH(CH₂)_(p)NHC(O)—, wherein p is an integer from about 2 to about20.
 20. The method of claim 19 wherein the polymer is furthercharacterized by a difunctional cross-linking monomer.
 21. The method ofclaim 20 wherein the difunctional cross-linking monomer is selected fromthe group consisting of diacrylates, triacrylates and tetraacrylates,dimethacrylates, diacrylamides, diallylacrylamide, di(methacrylamides),triallylamine and tetraalylammoniumion.
 22. The method of claim 21wherein the difunctional cross-linking monomer is selected from thegroup consisting of ethylene glycol diacrylate, propylene glycoldiacrylate, butylene glycol diacrylate, ethylene glycol dimethacrylate,butylene glycol dimethacrylate, methylene bis(methacrylamide), ethylenebis(acrylamide), ethylene bis(methacrylamide), ethylidenebis(acrylamide), ethylidene bis(methacrylamide), pentaerythritoltetraacrylate, trimethylolpropane triacrylate, bisphenol Adimethacrylate, bisphenol A diacrylate and divinylbenzene.
 23. A methodfor inhibiting a pathogenic toxin in a mammal, comprising the step ofadministering to the mammal a therapeutically effective amount of across-linked polymer, wherein said cross-linked polymer comprises firstand second polymer strands connected by a linking group, wherein: saidfirst polymer strand is characterized by a repeat unit having a primaryamino group or a secondary amino group; and said second polymer strandis characterized by a repeat unit having a tertiary amino group or aquaternary ammonium group.
 24. The method of claim 23 wherein the secondpolymer strand is further characterized by a repeat unit having primaryor secondary amino groups.
 25. The method of claim 23 wherein the firstpolymer strand is selected from the group consisting of polyallylamine,polyvinylamine, poly(ethyleneimine), polydiallylamine,poly(N-alkylallylamine), and poly(N-alkylvinylamine).
 26. The method ofclaim 25 wherein the first polymer strand is poly(N-methylallylamine) orpoly(N-methylvinylamine).
 27. The method of claim 24 wherein the secondpolymer strand is a copolymer characterized by a first repeat unitselected from the group consisting of N-alkyldiallylamine,N,N-dialkylallylamonium A-, N,N-dialkylallylamine, andN,N,N-trialkylallylammonium A-, wherein A- is an anion, and a secondrepeat unit selected from the group consisting of allylamine,vinylamine, diallylamine, N-alkylallylamine and N-alkylvinylamine. 28.The method of claim 27 wherein the second polymer strand ispoly(N-alkyldiallylamine-co-diallylamine);poly(N,N-dialkyldiallylamonium-co-allylamine) A-;poly(N,N-dialkylallylamine-co-allylamine);poly(N,N-dialkylallylamine-co-N-alkylallylamine);poly(N,N,N-trialkylallylammonium-co-allylamine) A-;poly(N,N,N-trialkylallylammonium-co-N-alkylallylamine) A-;poly(N,N-dialkylvinylamine-co-vinylamine);poly(N,N-dialkylvinylamine-co-N-alkylvinylamine);poly(N,N,N-trialkylvinylammonium-co-vinylamine) A-; orpoly(N,N,N-trialkylvinylammonium-co-N-alkylvinylamine) A-.
 29. Themethod of claim 23 wherein the first polymer strand and the secondpolymer strand are connected by a bridging group selected from the groupconsisting of straight chain or branched, substituted or unsubstitutedalkylene groups, diacylalkylene groups, diacylarene groups and alkylenebis(carbamoyl) groups.
 30. The method of claim 29 wherein the bridgingunit is selected from the group consisting of —(CH₂)_(n)—, wherein n isan integer from about 2 to about 20; —CH₂—CH(OH)—CH₂—; —C(O)CH₂CH₂C(O)—;—CH₂—CH(OH)—O—(CH₂)_(m)—O—CH(OH) —CH₂—, wherein m is 2 to about 4;—C(O)—(C₆H₂(COOH)₂)—C(O)—; and —C(O)NH(CH₂)_(p)NHC(O)—, wherein p is aninteger from about 2 to about
 20. 31. The method of claim 23 wherein thecross-linked polymer is produced by a method comprising the step ofcontacting a mixture comprising the first linear polymer and the secondlinear polymer with a crosslinking agent having two or more functionalgroups which react with amine groups to form a covalent bond, underconditions sufficient for cross-linking of the first linear polymer andthe second linear polymer.
 32. The method of claim 31 wherein thecrosslinking agent is selected from the group consisting ofepihalohydrin, succinyl dichloride, butanedioldiglycidyl ether,ethanedioldiglycidyl ether, pyromellitic dianhydride, dihaloalkanes anda,w-alkylene diisocyanates.
 33. The method of claim 1 wherein thepathogenic toxin is produced by one or more bacteria.
 34. The method ofclaim 33 wherein the one or more bacteria is selected from the groupconsisting of E. coli or C. difficile.
 35. The method of claim 1 whereinthe pathogenic toxin is produced by one or more viruses.
 36. The methodof claim 1 wherein the pathogenic toxin is produced by one or moreprotozoa.
 37. The method of claim 1 wherein the pathogenic toxin isproduced by one or more fungus.